LIISRARY 


UNIVERSITY  OF  CALIFORNIA, 


Class 


"BIOLOGY 


iamumtiHiu 


WORKS  BY 
CHARLES    F.    BOLDUAN,    M.D. 

PUBLISHED  BY 

JOHN  WILEY  &  SONS 


Immune  Sera. 

Antitoxins,  Agglutining,  Hsemolysins,  Bacterio- 
lysins,  Precipitins,  Cytotoxins,  and  Opsonins. 
New  edition,  rewritten.  By  Charles  F.  Bolduan, 
M.D.  12mo,  viii  +  176  pages.  Cloth,  $1.50. 

TRANSLATIONS. 

The  Suppression  of  Tuberculosis. 

Together  with  Observations  concerning  Phthisio- 
genesis  in  Man  and  Animals,  and  Suggestions  con- 
cerning the  Hygiene  of  Cow  Stables  and  the  Pro- 
duction of  Milk  for  Infant  Feeding,  with  Special 
Reference  to  Tuberculosis.  By  Professor  E.  von 
Behring,  University  of  Marburg.  Authorized 
Translation  by  Charles  F.  Bolduan,  M.D.  12mo, 
vi  +  85  pages.  Cloth,  SI. 00. 

Manual  of  Serum  Diagnosis. 

By  Doctor  O.  Rostoski,  University  of  Wurzburg. 
Authorized  Translation  by  Charles  F.  Bolduan, 
M.D.  12mo,  vi  + 80  pages.  Cloth,  $1.00. 

Collected  Studies  on  Immunity. 

By  Professor  Paul  Ehrlich.  Translated  by  Charles 
F.  Bolduan,  M.D.  8vo,  xi  +  586  pages.  Cloth, 
$6.00. 


IMMUNE  SERA 

A    CONCISE    EXPOSITION     OF     OUR    PRESENT    KNOWLEDGE 

CONCERNING    THE    CONSTITUTION    AND 

MODE    OF    ACTION    OF 

ANTITOXINS,  AGGLUTININS,  H^EMOLYSINS, 

BACTERIOLYSINS,    PRECIPITINS, 

CYTOTOXINS,    AND 

OPSONINS 


BY 
DR.  CHARLES    FREDERICK   BOLDUAN 

Bacteriologist,  Research  Laboratory,  Department  of  Health, 
City  of  New  York 


THIRD  EDITION,  ENLARGED 
FIRST    THOUSAND 


NEW  YORK 
JOHN    WILEY    &   SONS 

LONDON;   CHAPMAN   &   HALL,  LIMITED 

1908 


r* 

I    UNI 


OF  THE 

UNIVERSITY 

OF 


COPYRIGHT,  1907,  1908, 
BY  CHARLES  FREDERICK   BOLDUAN 


ttftr  Jlrroa 
fiuhrrt  DruntHtnuh  and  Olomftang 
i\*rto  tyark 


PREFACE 


THIS  book  has  its  origin  in  a  monograph  by 
Professor  Wassermann,  a  translation  of  which  was 
published  by  the  author  in  1904  under  the  title 
"  Immune  Sera."  While  much  of  the  material  con- 
tained in  that  book  will  be  found  in  the  present 
volume,  it  has  been  deemed  necessary  to  discuss 
more  fully  the  original  topics,  and  to  widen  the 
scope  of  the  book  by  adding  chapters  on  snake 
venoms  and  their  antisera,  agglutinins,  opsonins, 
and  serum  sickness.  The  author  gratefully  ac- 
knowledges his  indebtedness  to  Dr.  W.  H.  Park  for 
valuable  suggestions  in  the  preparation  of  the  book. 

NEW  YORK,  Sept.  i,  1907. 


NOTE  TO   THIRD   EDITION 

IN  response  to  a  number  of  inquiries,  the  author 
has  added  a  chapter  dealing  with  the  serum  diagno- 
sis of  syphilis.  This  concerns  mainly  the  test  de- 
vised by  Wassermann  and  subsequently  modified 
by  various  authors,  though  a  brief  description  of 
the  Porges-Meier  test  is  also  included.  Through 
the  courtesy  of  Dr.  Noguchi,  the  author  is  also 
able  to  present  a  note  on  a  simple,  as  yet  unpub- 
lished, test  devised  by  that  investigator. 

NEW  YORK,  July  15,  1908. 

174998 


CONTENTS 

PAGE 

Antitoxins  ..-.•,-. •   •  I 

HISTORICAL i 

PRESENT    METHOD    OF    PRODUCING    DIPHTHERIA    ANTI- 
TOXIN   2 

PRODUCTION  OF  DIPHTHERIA  TOXIN 2 

IMMUNIZING  THE  ANIMALS 3 

COLLECTING  THE  SERUM 4 

TESTING  THE  STRENGTH  OF  THE  SERUM      5 

EHRLICH'S  THEORY  FOR  PRODUCTION 6 

TOXINS,  TOXOIDS 6 

RECEPTORS      Q 

WEIGERT'S  OVERPRODUCTION  THEORY      10 

EXPERIMENTAL  EVIDENCE   FOR  EHRLICH'S  THEORY  13 

ANTIGENS  OR  HAPTINS 16 

NATURE  OF  ANTITOXINS  IN  GENERAL 17 

TOXINS    AND    OTHER    POISONOUS    CELL     DERIVATIVES     IN 

GENERAL 19 

RELATIONS  BETWEEN  TOXIN  AND  ANTITOXIN     ....  21 

"  L0  "  and  "  Lf  " 23 

PARTIAL  SATURATION  METHOD  OF  STUDYING  TOXINS 

-  TOXONS,  TOXOIDS 23 

EHRLICH'S   "  POISON  SPECTRA" 24 

VIEWS  OF  ARRHENIUS,   BORDET,  AND  OTHERS     ...  28 

Agglutinins 30 

THE   PHENOMENON 30 

PURPOSE  OF  AGGLUTINATION 33 

HISTORICAL 33 

PFAUNDI.ER'S  REACTION   (THREAD  REACTION)     .    „    „    .  35 

v 


VI  CONTENTS 

PAGE 

NATURE  OF  THE  AGGLUTININS 35 

NATURE  OF  THE  AGGLUTINATION  REACTION 36 

AGGLUTINOIDS 38 

GROUP  AGGLUTININS  — 39 

ABSORPTION  METHODS  FOR  DIFFERENTIATING  BETWEEN 

A  MIXED  AND  A  SINGLE  INFECTION 41 

FORMATION  OF  AGGLUTININS  ACCORDING  TO  THE  SIDE- 
CHAIN  THEORY,  RECEPTORS  OF  FIRST,  SECOND,  AND 

THIRD  ORDER 43 

Bacteriolysins  and  Hsemolysins 47 

HISTORICAL 47 

PFEIFFER'S  PHENOMENON 48 

HAEMOLYSIS 49 

NATURE  OF  H^EMOLYTIC  SERA 51 

THE  EXCITING  AGENT      54 

RESUME 54 

ANALOGY   BETWEEN  THE   BACTERIOLYTIC   AND   H.EMO- 

LYTIC  PROCESSES 54 

EHRLICH  AND  MORGENROTH  ON  THE  NATURE  OF  HAEMO- 
LYSIS       56 

THEIR  THREE  CLASSIC  EXPERIMENTS 57 

NOMENCLATURE 60 

ROLE  OF  THE  IMMUNE  BODY 62 

ON  WHAT  THE  SPECIFICITY  DEPENDS 63 

DIFFERENCE  BETWEEN  A  SPECIFIC  SERUM  AND  A  NOR- 
MAL ONE. 64 

DIVERGING  VIEWS  OF  EHRLICH  AND  BORDET     ....  64 

THE  SIDE-CHAIN   THEORY  APPLIED  TO  THESE   BODIES.  65 

MULTIPLICITY  OF  COMPLEMENTS 67 

THE      BORDET-GENGQU       PHENOMENON  ;       NEISSER- 

SACHS  BLOOD  TEST       68 

NORMAL  SERUM,  ITS.  H^MOLYTIC   AND   BACTERIOLYTIC 

ACTION         70 

ACTIVE  AND  INACTIVE  NORMAL  SERUM 72 


CONTENTS  vii 

PAGE 

ACTION  NOT  ENTIRELY  SPECIFIC 74 

MULTIPLICITY  OF  THE  ACTIVE  SUBSTANCES     ....  75 
DIFFERENCE  BETWEEN  A  NORMAL  AND  A  SPECIFIC  IM- 
MUNE SERUM 76 

NATURE    OF    THE    IMMUNE    BODY  —  PARTIAL    IMMUNE 

BODIES  OF  EHRLICH 79 

METCHNIKOFF'S  VIEWS 81 

SUPPORT  FOR  EHRLICH'S  VIEW 82 

ANTIH^EMOLYSINS:  THEIR  NATURE  —  ANTI-COMPLEMENT 

OR  ANTI-IMMUNE  BODY 84 

ANTI-COMPLEMENT 85 

ANTO-ANTICOMPLEMENTS  .    . 88 

FLUCTUATIONS  IN  THE  AMOUNT  OF  THE  ACTIVE  SUB- 
STANCES IN  SERUM , 90 

SOURCE    OF    THE    COMPLEMENTS  —  LEUCOCYTES    AS    A 

SOURCE  —  OTHER  SOURCES 92 

STRUCTURE  OF  THE  COMPLEMENTS  —  COMPLEMENTOIDS  93 

ISOLYSINS  AUTOLYSINS  ANTI-ISOLYSINS    .....  95 

DEFLECTION  OF  COMPLEMENT 97 

DEUTSCH'S  H^EMOLYTIC  BLOOD  TEST 102 

PRACTICAL  VALUE  OF  BACTERICIDAL  SERA 104 

Precipitins 106 

DEFINITIONS .  106 

BACTERIAL  PRECIPITINS 107 

LACTOSERUM  —  OTHER  SPECIFIC  PRECIPITINS     ....  107 

SPECIFICITY  OF  THE  PRECIPITINS „    .    .    .  108 

NATURE  OF  THE  PRECIPITINS no 

PRACTICAL  APPLICATION ,    ,    .  m 

THE  WASSERMANN-UHLENHUTH  BLOOD  TEST     ...  112 

IMMUNIZING  THE  ANIMALS 113 

COLLECTING  THE  SERUM 114 

THE  TEST 115 

APPEARANCE  OF  THE  REACTION 116 

DELICACY  OF  THE  PRECIPITIN  TEST     «,,,,.  117 


viii  CONTENTS 

PAGB 

OTHER  APPLICATIONS  OF  THE  PRECIPITIN  TEST     .    .  117 

ANTIPRECIPITINS  —  ISOPRECIPITINS    .    ...    .......  118 

Cytotoxins 119 

DEFINITION,  LEUCOTOXIN  — NATURE  OF  THE  CYTOTOXIN 

ANTICYTOTOXIN 119 

NEUROTOXIN 120 

SPERMOTOXIN 121 

COMMON  RECEPTORS. 122 

CYTOTOXIN  FOR  EPITHELIUM 122 

CYTOTOXINS  BY  THE  USE  OF  NUCLEOPROTEIDS       ...  123 

Opsonins 125 

HISTORICAL.    . ...... 125 

BACTERIOTROPIC  SUBSTANCES •   •  «  127 

OPSONINS  DISTINCT  ANTIBODIES „    .    .    .  128 

STRUCTURE  OF  OPSONINS     .....    0 128 

THE  OPSONIC  INDEX „ 128 

TECHNIQUE 129 

VALUE  OF  THE  OPSONIC  MEASUREMENTS 132 

Snake  Venoms  and  their  Antisera  ........  135 

THE  VENOMS  .    .    .    .    , „ 135 

ANTIVENINS 137 

Serum  Sickness 138 

DEFINITION. .    .    .    .    „ 138 

DUE  TO  SERUM  AS  SUCH 139 

VON  PlRQUET   AND  SCHICK'S  THEORY       ,, 140 

ANAPHYLAXIS  .....................  142 

THE  CONCENTRATION  AND  PURIFICATION  OF  ANTITOXIC 

SERA 145 

Appendices , 147 

A    SERUM  DIAGNOSIS  OF  SYPHILIS 147 

B.  NOGUCHI'S  BUTYRIC  ACID  TEST 164 


IMMUNE  SERA 


ANTITOXINS 

Historical. -- The  researches  of  Buchner1  in  1889 
had  shown  that  the  serum  of  animals  artificially 
immunized  against  a  certain  bacterium  possessed 
marked  bactericidal  properties  for  that  particular 
organism.  In  studying  immunity  on  animals  which 
had  been  successfully  immunized  against  diphtheria 
infection,  Behring,2  working  in  Koch's  laboratory 
was  struck  by  the  fact  that  in  these  animals 
living  virulent  diphtheria  bacilli  were  often  demon- 
strable in  the  scab  at  the  site  of  injection  several 
weeks  after  the  infection,  and  furthermore  that  the 
blood  serum  of  the  animals  did  not  possess  bacteri- 
cidal properties.  In  a  study  published  in  1890 
Behring  showed  that  the  serum  of  rabbits  arti- 
ficially immunized  against  diphtheria  was  able  to 
confer  a  specific  immunity  against  diphtheria  infec- 
tions in  other  animals.  He  also  demonstrated  that 
such  a  serum  could  be  used  therapeutically  to  cure 
an  infection  already  in  progress.  Such  a  serum 

1  Buchner,  Centralblatt  Bacteriologie,  Vol.  v.  1889.     Archiv. 
f.  Hygiene,  Vol.  x.  1890. 

2  Behring    &    Kitasato,    Deutsche    med.    Wochenschrift,  No. 
49,     1890. 


2  IMMUNE  SERA 

was  not  bactericidal,  and  retained  its  therapeutic 
power  for  a  considerable  time.  He  believed  that 
the  action  of  the  serum  was  effected  by  a  neu- 
tralization of  the  bacterial  toxin  by  an  "  antitoxic 
serum  constituent. ' '  The  action  was  strictly  specific, 
an  antitoxic  serum  obtained  after  a  diphtheria 
infection  protected  only  against  diphtheria;  one 
derived  from  a  tetanus  animal,  only  against  tetanus. 
Subsequently  Behring  and  Knorr  showed  that  im- 
munization could  be  effected  with  bacterial-free 
filtrates  of  tetanus  cultures  and  that  the  serum  thus 
produced  protected  not  only  against  tetanus 
infection  but  against  poisoning  by  the  toxic  prod- 
ucts of  the  bacilli.  After  considerable  experi- 
mental work  Behring  and  his  collaborators  devised 
an  effective  method  of  immunizing  ^heep  and 
certain  other  animals  against  diphtheria  and  against 
tetanus  and  so  produced  antitoxic  sera  in  con- 
siderable amounts. 

The  following  account  taken  from  Park  shows  the 
present  methods  of  producing  diphtheria  antitoxin. 

Production  of  the  Diphtheria  Toxin.  — A  strong 
diphtheria  toxin  should  be  obtained  by  taking  a  very 
,*q.rulent  culture  and  growing  it  in  broth  which  is  about 
/  8  cc.  normal  soda  solution  per  liter  above  the  neutral 
point  to  litmus."")  The  culture  fluid  should  be  in  com- 
paratively thin"  layers  and  in  large-necked  Erlenmeyer 
flasks,  so  as  to  allow  of  a  free  access  of  air;  the  tem- 
perature should  be  about  35°  to  36°  C.  The  culture, 
after  a  weeks  growth,  is  removed  from  the  incubator, 


ANTITOXINS  3 

and  having  been  tested  for  purity  by  microscopic  and 
culture  tests  is  rendered  sterile  by  the  addition  of  10 
per  cent  of  a  5  per  cent  solution  of  carbolic  acid.  After 
48  hours  the  dead  bacilli  have  settled  on  the  bottom  of 
the  jar  and  the  clear  fluid  is  filtered  through  ordinary 
sterile  filter  paper  and  stored  in  full  bottles  in  a 
cold  place  until  needed.  Its  strength  is  then  tested  by 
giving  a  series  of  guinea  pigs  carefully  measured 
amounts.  Less  than  o.oi  cc.  when  injected  hypoder- 
mically  should  kill  a  250  gram  guinea  pig. 

Immunizing  the  Animals.  — The  horses  used  should 
be  young,  vigorous,  of  fair  size,  and  absolutely  healthy. 
Vicious  habits,  such  as  kicking,  etc.,  make  no  difference, 
except,  of  course,  to  those  who  handle  the  animals. 
The  horses  are  severally  injected  with  an  amount  of 
toxin  sufficient  to  kill  five  thousand  guinea  pigs  of  250 
grams  weight  (about  20  cc.  of  strong  toxin).  After 
from  three  to  five  days,  so  soon  as  the  fever  reaction 
has  subsided,  a  second  subcutaneous  injection  of  a 
slightly  larger  dose  is  given.  With  the  first  three 
injections  of  toxin  10,000  units  of  antitoxin  are  given. 
If  antitoxin  is  not  mixed  with  the  first  doses  of  toxin 
only  one-tenth  of  the  doses  advised  is  to  be  given. 
At  intervals  of  from  five  to  eight  days  increasing  injec- 
tions of  pure  toxin  are  made  until  at  the  end  of  two 
months  from  ten  to  twenty  times  the  original  amount 
is  given.  There  is  absolutely  no  way  of  judging  which 
horses  will  produce  the  highest  grades  of  antitoxin. 
Very  roughly  those  horses  which  are  extremely  sensi- 
tive, and  those  which  react  hardly  at  all  are  the  poorest, 
but  even  here  there  are  exceptions.  The  only  way, 
therefore,  is  at  the  end  of  six  weeks  or  two  months  to 
bleed  the  horses  and  test  their  serum.  If  only  high 
grade  serum  is  wanted  all  the  horses  that  give  less 


4  IMMUNE  SERA 

than  150  units  per  cc.  are  discarded.  If  moderate 
grades  only  are  desired,  all  that  yield  100  units  may  be 
retained.  The  retained  horses  receive  steadily  in- 
creasing doses,  the  rapidity  of  the  increase  and  the 
interval  of  time  between  the  doses  (three  days  to  one 
week)  depending  somewhat  on  the  reaction  following 
the  injection,  an  elevation  of  temperature  of  more  than 
3°  F.  being  undesirable.  At  the  end  of  three  months 
the  antitoxic  serum  of  all  the  horses  should  contain 
over  300  units  and  in  about  10  per  cent  as  much  as  800 
units  per  cc.  Very  few  horses  ever  give  over  1000 
units,  and  none  so  far  has  given  as  much  as  2000  units 
per  cc.  The  very  best  horses,  if  pushed  to  their 
limit  continue  to  furnish  blood  of  gradually  decreasing 
strength.  If  every  nine  months  an  interval  of  three 
months'  freedom  from  inoculations  is  given,  the  best 
horses  furnish  high  grade  serum  during  their  periods  of 
treatment  for  from  two  to  four  years. 

Collecting  the  Serum.  —  In  order  to  obtain  the  serum 
the  blood  is  withdrawn  from  the  jugular  vein  by  means 
of  a  sharp-pointed  canula  which  is  plunged  through  the 
vein  wall,  a  slit  having  been  made  in  the  skin.  The 
blood  is  carried  by  a  sterile  rubber  tube  attached  to  the 
canula,  into  large  Erlenmeyer  flasks  and  allowed  to 
clot,  the  flasks,  however  being  placed  in  a  slanting 
position  before  clotting  has  commenced.  The  serum 
is  drawn  off  after  4  days  by  means  of  sterile  glass  and 
rubber  tubing,  and  is  stored  in  large  flasks  in  a  refrige- 
rator. From  this  as  needed  small  vials  are  filled. 
The  vials  and  their  stoppers,  as  indeed  all  the  utensils 
used  for  holding  the  serum,  must  be  absolutely  sterile 
and  evqry  possible  precaution  must  be  taken  to  avoid 
contamination  of  the  serum.  An  antiseptic  may  be 
added  as  a  preservative,  but  is  not  necessary.  Diph- 


ANTITOXINS  5 

theria  antitoxin,  when  stored  in  vials  and  kept  in  a  cool 
place  away  from  light  and  air  contains  within  10  per 
cent  of  its  original  strength  for  at  least  two  months; 
after  that  it  can  be  used  by  allowing  for  a  maximum 
deterioration  of  3  per  cent  for  each  month. 

Testing  the  Strength  of  the  Antitoxin.  — This  is  carried 
out  as  follows:  Six  guinea  pigs  are  injected  with  mix- 
tures of  toxin  and  antitoxin.  In  each  of  the  mixtures 
there  is  100  times  the  amount  of  a  toxin  (similar  to 
that  adopted  as  the  standard)  which  will  kill  a  250 
grams  on  an  average  in  96  hours.  In  each  of  the 
mixtures  the  amount  of  antitoxin  varies;  for  instance, 
No.  i  would  contain  0.002  cc.  serum;  No.  2,  0.003  cc-> 
No.  3,  0.064  cc.;  No.  4,  0.005  cc->  etc-  If  at  the  en(^  °f 
the  fourth  day  Nos.  i,  2  and  3  were  dead  and  Nos.  4, 
5  and  6  were  alive  we  would  consider  the  serum  to 
contain  200  units  of  antitoxin  for  each  cubic  centi- 
meter. When  we  mix  only  ten  fatal  doses  of  toxin 
with  one-tenth  ok  the  amount  .of  antitoxin  used  with 
100  fatal  doses,  the  guinea  pig  must  remain  well.  The 
mixed  toxin  and  antitoxin  must  remain  together  for 
fifteen  minutes  before  injecting. 

Behring's  publication  was  followed  in  the  next 
two  years  by  considerable  work  along  these  lines, 
valuable  contributions  being  made  by  Aronson,1 
Roux,  and  Martin,2  Wernicke,3  Knorr 4  and  others. 
The  statements  of  Behring  as  to  the  strict  specifi- 
city of  the  antitoxins  were  fully  confirmed.  Certain 

1  Berliner  med.   Gesellschaft,  Sitzung,   Dec.   21,    1892.     Also 
Berliner  Klin.  Wochenschrift,  1893  and  1894. 

2  Roux  and  Martin,  Annal.  Pasteur    1894. 

3  Behring  and  Wernicke,  Zeitsch.   Hygiene,  1892.     Vol.  xi. 

4  Behring  and  Knorr,  Zeitsch.   f.  Hygiene,  1893.     Vol.  xii. 


6  IMMUNE  SERA 

observations  by  Buchner 1  and  by  Roux  and  Martin 
threw  doubt,  however,  on  the  correctness  of  Beh- 
rings  view  that  the  toxin  was  neutralized  by  the 
specific  serum  just  as  a  base  was  neutralized  by  an 
acid.  It  was  claimed,  for  example,  that  the  specific 
serum  acted  mainly  on  the  body  cells  causing  them 
to  become  non-susceptible  to  the  poison  in  question. 
Various  theories  were  formulated  to  account  for  the 
production  of  the  antitoxins,  their  specificity,  etc., 
but  of  them  all  only  one  has  at  all  maintained  itself. 
This,  is  the  so-called  side-chain  theory,  which  was 
formulated  by  Ehrlich2  in  1897. 

Ehrlich's  Side-Chain  Theory.  —  Originally  the 
side-chain  theory  was  applied  by  Ehrlich  only  to 
the  production  of  the  specific  antitoxins,  i.e.,  sub- 
stances in  the  blood,  which  act  not  only  on  the 
living  bacteria,  but  also  and  especially  on  their 
dissolved  toxins.  Later  on  he  extended  it  so  as 
to  apply  also  to  the  formation  of  specific  bacteri- 
cidal and  hasmolytic  substances  in  the  serum  of 
animals  treated  with  living  bacteria  or  with  animal 
cells. 

Toxins  —  -  Toxoids  —  Special  Function  of  the  Side 
Chains.  -  -  The  basis  of  the  theory  is  the  fact  that 
poison  and  counter-poison,  toxin  and  antitoxin, 
combine  directly  in  any  given  quantity.  This 
combination  always  occurs  in  definite  proportions 

1  Buchner,  Miinchener  med.  Wochenschrift,  1894. 
*  Ehrlich,  Klinisches  Jahrbuch,  1897. 


ANTITOXINS  7 

following  the  laws  of  chemical  combination;  and, 
still  following  those  laws,  is  slower  at  lower  tem- 
peratures than  at  higher,  stronger  in  concentrated 
than  in  dilute  form.  Ehrlich  could  further  show 
that  each  poison  for  which  by  the  process  of  immun- 
izing one  can  develop  a  counter-poison  possesses 
two  groups  which  are  concerned  in  the  combina- 
tion with  the  counter-poison  or  antitoxin.  One  of 
these,  the  so-called  haptophore  group,  is  the  combin- 
ing group  proper;  the  other,  the  toxophore  group, 
is  the  carrier  of  the  poison.  A  poison  molecule, 
therefore,  might  lose  the  one,  the  toxophore,  and 
still  be  capable  by  means  of  its  haptophore  group 
of  combining  with  antitoxin.  Such  a  modified 
poison,  which  because  of  the  loss  of  the  toxophore 
group  can  hardly  be  called  a  poison,  but  which  still 
possesses  the  power  to  combine  with  antitoxin, 
Ehrlich  calls  a  toxoid.  Toxoids  may  be  produced 
spontaneously  in  old  poisons  through  decomposi- 
tion of  the  poison  molecule,  or  they  may  be  pro- 
duced artificially  by  causing  certain  destructive 
agents  such  as  heat  or  chemicals  to  act  on  bacterial 
poisons.  The  toxophore  group  is  a  very  delicate 
one  and  much  more  readily  decomposed  than  the 
combining  (haptophore)  group.  Ehrlich  reasoned 
that  in  order  for  a  poison  to  be  toxic  to  an  organ- 
ism, i.e.,  in  order  that  the  toxophore  group  be  able 
to  act  destructively  on  a  cell,  it  is  necessary  for  the 
haptophore  group  of  the  poison  to  combine  with 


8 


IMMUNE   SERA 


the  cell.  "  In  every  living  cell,"  Ehrlich  says, 
"  there  must  exist  a  dominating  body  [Leistungs 
Kern]  and  a  number  of  other  chemical  groups  or 
side  chains.  These  groups  have  the  greatest  variety 
-of  function,  but  especially  those  of  nutrition  and 
assimilation." 

The  side  chains,  then,  according  to  this  author, 


toxophore  group 


,   POISON   MOLECULE 


FlG.  I 

are  able  to  combine  with  the  greatest  variety  of 
foreign  substances  and  convert  these  into  nourish- 
ment suitable  to  the  requirements  of  the  active 
central  body.  They  are  comparable  to  the  pseudo- 
podia  of  the  lower  animals,  which  engulf  food  par- 
ticles and  assimilate  the  same  for  the  immediate 
use  of  the  organism.  In  order  that  any  substance 


ANTITOXINS  9 

may  combine  with  these  side  chains  it  is  necessary 
that  certain  very  definite  relations  exist  between 
the  combining  group  of  the  substance  and  that 
of  the  side  chain.  Using  the  well-known  simile  of 
Emil  Fischer,  the  relation  must  be  like  that  of  lock 
and  key,  i.e.,  the  two  groups  must  fit  accurately. 
Hence  not  every  substance  will  fit  all  the  side 
chains  of  an  organism.  It  will  combine  only  with 
those  for  which  is  possesses  a  fitting  group. 

Receptors  —  Weigerfs  Overproduction  Theory.  — 
This  doctrine  of  the  chemistry  of  the  organism's 
metabolism  Ehrlich  applied  to  the  action  of  toxins 
and  antitoxins.  "  The  toxin,"  he  said,  "  can  act 
only  when  its  haptophore  group  happens  to  fit  to 
one  of  the  side  chains,"  or  receptors,  as  he  now  pre- 
fers to  call  them.  As  a  result  of  this  combination, 
the  toxophore  group  is  able  to  act  on  the  cell  and 
injure  it.  If  we  take  as  an  example  tetanus,  in 
which  all  the  symptoms  are  due  to  the  central  ner- 
vous system,  the  side-chain  theory  assumes  that 
the  haptophore  group  of  the  tetanus  poison  fits 
exactly  and  is  combined  with  the  side  chain  or 
receptors  of  the  central  nervous  system.  Other 
experiments,  which  we  will  not  reproduce  here, 
have-  shown  us  unquestionably  that  the  action  of 
the  antitoxins  depends  on  the  fact  that  this  com- 
bines with  the  haptophore  group  of  the  poison  and 
so  satisfies  the  latter's  affinity.  Ehrlich,  therefore, 
concluded  that  the  antitoxin  is  nothing  else  than 


10  IMMUNE  SERA 

the  side  chains  or  receptors  which  are  given  off  by 
the  cells  and  thrust  into  the  circulation.  The  way 
in  which  these  side  chains  or  receptors  are  thrust 
off  as  a  result  of  the  immunizing  process,  Ehrlich 
explains  by  means  of  Weigert's  Overproduction 
Theory. 

At  the  meeting  of  German  Naturalists  and 
Physicians  held  at  Frankfurt  in  1896,  Weigert l  in 
discussing  regeneration,  advanced  an  hypothesis  the 
essential  features  of  which  are  that  physiological 
structure  and  function  depend  upon  the  equilibrium 
of  the  tissues  maintained  by  virtue  of  mutual 
restraint  between  their  component  cells ;  that  destruc- 
tion of  a  single  integer  or  group  of  integers  of  a 
tissue  or  a  cell  removes  a  corresponding  amount  of 
restraint  at  the  point  injured,  and  therefore  destroys 
equilibrium  and  permits  of  the  abnormal  exhibi- 
tion of  bioplastic  energies  on  the  part  of  the  remain- 
ing uninjured  components,  which  activity  may  be 
viewed  as"  a  compensating  hyperplasia;  that  hyper- 
plasia  is  not,  therefore,  the  direct  result  of  external 
irritation,  and  cannot  be,  since  the  action  of  the 
irritant  is  destructive  and  is  confined  to  the  cells 
or  integers  of  cells  that  it  destroys,  but  occurs 
rather  indirectly  as  a  function  of  the  surrounding 
uninjured  tissues  that  have  been  excited  to  bio- 
plastic  activity  through  the  removal  of  the  restraint 

1  Weigert,  Verhandlungen   der  Ges.  deutscher  Naturforscher 
iind  Aerzte,  1896. 


ANTITOXINS  1 1 

hitherto  exerted  by  the  cells  destroyed  by  the 
irritant;  and,  finally,  when  such  "bioplastic  activity 
is  called  into  play  there  is  always  hypercompen- 
sation  —  i.e.  there  is  more  plastic  material  gene- 
rated than  is  necessary  to  compensate  for  the 
loss. 

Ehrlich  points  out  that  owing  to  the  combination 
of  the  toxin  with  the  side  chain  of  a  cell,  these 
side  chains  are  practically  lost  to  the  cell;  that  the 
latter  or  its  fellows  now  produces  new  side  chains  to 
replace  this  loss,  but  that  this  production  always 
goes  so  far  as  to  make  a  surplus  of  side  chains ;  that 
these  side  chains  are  thrown  off  by  the  cell  as 
unnecessary  ballast,  and  then  circulate  in  the  blood 
as  antitoxin.  The  same  substances,  therefore,  which 
when  part  of  the  cell  combine  with  the  haptophore 
group  of  the  toxin,  enabling  that  to  act  on  the  cell, 
when  circulating  free  in  the  blood  combine  with 
and  satisfy  this  haptophore  group  of  the  toxin, 
and  prevent  the  poison  from  combining  with  and 
damaging  the  cells  of  the  organism. 

It  does  not  follow  from  Ehrlich's  theory  that  the 
antitoxin  is  produced  by  the  same  set  of  cells  whose 
injury  by  the  toxin  gives  rise  to  the  particular 
clinical  symptoms.  Thus  we  might  believe  that 
although  in  tetanus  the  cells  of  the  central  nervous 
system  give  rise  to  the  characteristic  symptoms,  cells 
entirely  apart  from  these,  e.g.,  in  the  bone  marrow, 
might  be  the  main  source  of  the  antitoxin.  The 


12  IMMUNE  SERA 

fact  that  we  appreciate  symptoms  from  only  one 
organ  is,  obviously,  no  proof  that  other  tissues 
have  been  unaffected. 

It  may  be  well  here  to  call  attention  to  another 
rather  common  misconception  regarding  the  pro- 
duction .  of  antitoxin,  namely  that  the  body  cells 
have  to  become  educated,  so  to  speak,  to  produce 
the  antitoxin.  This,  it  is  believed,  is  effected  by 
giving  gradually  increasing  doses  of  toxin.  As  a 
matter  of  fact  the  reason  for  this  gradual  increase 
in  the  dose  injected  is  quite  different.  The  object 
in  view  is  the  administration  of  an  enormously 
large  dose  of  toxin,  one  that  will  engage  the  recep- 
tors of  many  cells.  The  previous  injections  have 
brought  about  some  production  of  antitoxin  and 
this  partially  neurtalizes  some  of  the  toxin  in- 
jected, making  it  possible  to  give  a  larger  dose  than 
before.  If  one  gives  at  the  outset  a  large  amount  of 
toxin,  partially  neutralized  by  antitoxin,  one  will 
produce  an  amount  of  antitoxin  equal  to  that 
ordinarily  obtained  in  response  to  the  same  quan- 
tity of  unaltered  toxin  given  as  the  tenth  or 
twentieth  injection  of  a  series.  Park  and  Atkinson 
for  example,  injected  a  fresh  horse  with  one  litre 
of  a  toxin  neutralized  ij  times  for  guinea  pigs. 
At  the  end  of  a  week  the  horse  had  produced  a  serum 
containing  60  units  per  cc.  When  the  toxin  was 
neutralized  6  fold  no  antitoxin  whatever  was  pro- 
duced. 


ANTITOXINS  13 

Experimental  Evidence  for  Ehrlictis  Theory.  — 
According  to  Ehrlich,  then,  the  formation  of  specific 
antibodies  must  proceed  in  three  stages: 

1.  The  binding  of  the  haptophore  group  to  the 
receptor. 

2.  The    increased    production    of    the    receptors 
following  this  binding. 

3.  The  thrusting-off  of  these  increased  receptors 
into   the  blood. 

So  far  as  the  first  point  is  concerned  Wassermann  * 
showed  that  with  tetanus,  in  which,  as  is  well 
known,  all  the  symptoms  are  referable  to  the  cen- 
tral nervous  system,  tetanus  toxin  was  bound  by 
central  nervous  system  substance  in  vitro.  A 
mixture  of  tetanus  poison  and  normal  central 
nervous  system  was  innocuous  to  animals,  showing 
that  certain  substances  present  in  the  central 
nervous  system  combine  with  and  thus  satisfy  the 
affinity  of  the  haptophore  group  of  the  poison. 
This  of  course  prevents  the  latter  from  combining 
with  any  cells  of  the  organism.  Organs  other  than 
the  central  nervous  system  do  not  possess  this 
property  of  combining  with  tetanus  poison,  just 
as  the  central  nervous  system  is,  on  the  contrary, 
incapable  of  combining  with  diphtheria  poison, 
which  clinically  does  not  show  any  pronounced 
affinity  for  the  central  nervous  system. 

Wassermann  2  also  believes  recently  to  have  given 

1  Wassermann  and  Takaki,   Berliner  Klin.  Wochenschr,  1898. 
*  Wassermann,  New  York  Medical  Journal,  1904. 


14  IMMUNE  SERA 

experimental  proof  of  the  second  and  third  points, 
the  increased  production  of  the  receptors  and  their 
thrusting  off.  For  this  purpose  he  employed  a 
tetanus  poison  which  he  had  kept  for  about  eight 
years,  and  which  was  originally  very  poisonous. 
In  the  course  of  years,  however,  owing  to  the 
damaging  action  of  light,  of  oxidation,  etc.,  it  had 
become  so  weak  that  it  was  no  longer  toxic  at  all. 
Injections  of  one  cc.  into  a  guinea  pig  produced 
no  tetanus.  Nevertheless  the  haptophore  group 
remained  intact,  as  could  readily  be  proved,  for 
this  non-poisonous  tetanus  toxin  was  still  able  to 
bind  tetanus  antitoxin,  i.e.  thrust-off  receptors.  On 
injecting  rabbits  with  this  non-poisonous  tetanus 
toxoid  in  increasing  doses,  and  then  examining  the 
blood  serum  of  the  animal  he  found  not  a  trace  of 
tetanus  antitoxin.  This  absence  could  have  either 
of  two  causes:  It  might  be  that  the  toxoid  no 
longer  produced  any  physiological  effect  whatever 
in  the  organism;  or  although  it  still  caused  an 
increase  in  the  receptors,  these  increased  receptors 
remained  in  the  organs  (sessile)  and  were  not 
thrust  off  into  the  blood.  In  order  to  decide  this 
question  Wassermann  first  determined  the  exact 
quantity  of  fresh  tetanus  toxin  which  constituted  a 
fatal  dose  for  guinea  pigs.  He  reasoned  that  if 
he  injected  first  the  toxoid,  and  shortly  after,  say 
in  one  or  two  hours,  the  fresh  toxin,  he  should  in 
such  an  animal  have  to  increase  the  fatal  dose, 


ANTITOXINS  15 

i.e.  more  tetanus  toxin  should  be  required  to  kill 
this  animal  than  a  normal  one,  because  owing  to 
the  previous  toxoid  injection  part  of  the  cells  sus- 
ceptible to  tetanus  toxin  would  already  have  been 
occupied.  Provided  Ehrlich's  theory  were  correct, 
so  that  this  binding  of  the  toxoid  really  occurred, 
the  conditions  should  be  entirely  different  when, 
instead  of  injecting  the  toxin  shortly  after  the 
toxoid,  he  waited  somewhat  longer,  one  to  three 
days,  and  then  injected  the  fresh  tetanus  toxin0 
In  that  case  Weigert's  law  should  come  into  play 
and  the  receptors  have  commenced  to  increase 
in  number,  i.e.  the  organ  should  now  possess  more 
sensitive  groups  than  before.  This  would  manifest 
itself  in  such  fashion  that  in  contrast  to  the  first 
experiment  the  fatal  dose  of  fresh  tetanus  toxin 
could  now  be  decreased ;  in  other  words  a  small  dose 
would  now  tetanize  the  animal  in  a  shorter  time. 

As  a  matter  of  fact  Wassermann's  experiments 
yielded  exactly  the  results  deduced  theoretically. 
He  injected  a  guinea  pig  with  some  of  the  non- 
poisonous  toxoid  and  then,  an  hour  later,  with 
tetanus  toxin.  He  found  that  much  more  toxin 
was  required  to  kill  this  animal  than  a  normal 
guinea  pig*bf  equal  size.  When,  on  the  contrary, 
he  waited  one  to  three  days,  it  was  found  that  then 
a  dose  of  tetanus  toxin  which  would  not  even 
tetanize  a  normal  guinea  pig  was  sufficient  to  kill 
this  one. 


16  IMMUNE  SERA 

It  will  be  seen  that  in  the  above  experiments 
the  completely  non-poisonous  toxoid,  although  it 
effected  an  increased  production  of  receptors,  did 
not  cause  their  thrusting-off.  The  serum  of  the 
rabbit  treated  with  toxoid  contained  no  antitoxin 
whatever.  Wassermann  concludes  from  this  and 
other  experiments  that  the  thrusting-off  cannot  be 
a  function  of  the  haptophore  group,  and  that 
something  additional  is  required.  This  "  some- 
thing," he  claims  is  a  function  of  the  toxophore 
group.  It  may  be  stated  that  Von  Dungern  has 
also  published  experiments  (with  majaplasm)  point- 
ing to  the  existence  of  the  second  stage,  the  stage  of 
sessile  receptors. 

Antigens  or  Haptins.  —  It  has  been  found  that 
it  is  impossible  to  produce  any  immunity  against 
all  poisons,  e.g.  strychnine  or  morphine.  Accord- 
ing to  Ehrlich  these  simpler  chemical  molecules  do 
not  enter  into  a  true  chemical  combination  with 
the  tissues,  but  form  rather  a  kind  of  solid  solution, 
a  loose  combination  with  the  cells,  so  that  they  can 
again  be  abstracted  from  these  cells  by  all  kinds  of 
solvents,  e.g.  by  shaking  out  with  ether  or  chloro- 
form. The  point  can  perhaps  be  likened  to  the 
difference  between  saccharin  and  sugar.  Both  sub- 
stances taste  sweet,  but  despite  this  similarity  in 
their  physiological  action  they  behave  very  dif- 
ferently toward  the  cells  of  the  organism.  Sac- 
charin simply  passes  through  the  organism  without 


ANTITOXINS  I/ 

entering  into  a  firm  combination,  i.e.  without  being 
assimilated,  and  is  therefore  no  food.  Its  sweeten- 
ing action  is  a  mere  contact  effect  on  the  cells 
sensitive  to  taste.  Sugar,  on  the  contrary,  is 
actually  bound  by  the  cells,  assimilated  and  burnt, 
and  so  is  a  true  food.  Until  recently  it  was  believed 
that  the  simpler  chemical  substances  could  not 
excite  the  production  of  antibodies.  Ford  and 
Abel l  have  however  been  able  to  show  that  toad 
stool  poison,  a  true  toxin,  against  which  an  anti- 
toxin can  be  produced  is  chemically  a  glucoside. 

As  we  shall  subsequently  see  it  is  possible  to 
immunize  the  animal  body  against  a  large  number 
of  substances,  including  not  only  such  cell  products 
as  ferments,  toxins  and  venoms,  but  also  cells  of 
the  greatest  variety,  bacteria,  dissolved  proteids,  etc. 
All  these  substances,  therefore,  must  possess  hapto- 
phore  groups  able  to  combine  with  the  side  chains 
or  receptors  in  the  animal  body.  Collectively, 
•we  speak  of  such  substances  as  antigens  or  haptins. 

Nature  of  Antitoxins  in  General.  —  But  little  is 
known  concerning  the  constitution  of  antitoxins, 
for  we  do  not  know  them  apart  from  serum  or 
serum  constituents.  It  seems  probable  that  they 
are  proteid  in  character,  but  this  has  not  been 
positively  decided.  It  has  been  found  that  like 
the  globulins  they  are  quite  resistant  to  the  action 
of  trypsin,  but  are  acted  on  by  pepsin-hydrochloric 

1  Ford  and  Abel.  Journal  of  Biological  Chemistry,  Vol.  ii,  1907. 


1 8  IMMUNE   SERA 

acid.  In  general  they  withstand  a  fair  degree  of 
heat,  certainly  far  more  than  the  toxins.  Anti- 
toxins are  to  be  regarded  as  inactive  substances, 
effecting  merely  a  blocking  of  the  haptophore 
group  of  the  corresponding  toxin.  They  do  not 
act  on  the  toxins  destructively.  This  is  indicated 
by  experiments  of  Wassermann  on  pyocyaneus  toxin, 
and  of  Calmette  and  Morgenroth  1  on  snake  venom, 
which  showed  that  in  the  toxin-antitoxin  com- 
bination, the  toxin  could  again  manifest  itself  after 
the  antitoxin  had  been  destroyed.  The  antitoxins 
therefore  are  not  ferment-like  substances.  As  far 
back  as  1897  attempts  were  made  to  determine  the 
chemical  nature  of  the  antitoxins.  In  that  year 
Belfanti  and  Carbone2  found  that  the  antitoxin 
was  precipitated  with  the  globulins  of  the  serum  by 
means  of  magnesium  sulphate.  Dieudonne 3  had 
previously  shown  that  the  proteids  thrown  out  of 
solution  by  acetic  and  carbonic  acids  contained 
none  of  the  antitoxin.  In  1901  Atkinson4  showed 
that  the  globulins  increase  markedly  in  the  serum 
of  horses  as  the  antitoxic  strength  increases.  The 
most  recent  work  on  this  subject  is  that  of  Gibson,5 
who  shows  that  if  the  ammonium  sulphate  precipi- 

1  Morgenroth,  Berlin,  klin.  Wochenschr.  1905. 

2  Beifanti    and    Carbone,    Centralblatt    Bacteriologie    (Ref.), 
Vol.  xxiii,  1898. 

3  Dieudonne,  Arbeiten  a.d.  kaiserl.     Gesundheitsamte.     Vol. 
xiii,  1897. 

4  Atkinson,  Jour.  Exper.  Medicine,  Vol.  i,  1901. 

5  Gibson,  Journ.  Biological  Chemistry,  Vol.  i,  1906, 


ANTITOXINS  19 

tate  (globulins,  nucleo-proteids,  etc.)  is  treated  with 
saturated  sodium  chloride  solution,  practically  all 
the  antitoxic  fraction  passes  into  solution. 

This  author  has  recently  studied  the  possibility 
of  differentiating  other  antibodies  by  means  of 
their  precipitation  characteristics.  He  believes  that 
a  differentiation  of  the  antibodies  into  those  pre- 
cipitated with  the  pseudo  globulins  and  with  the 
euglobulin  fractions,  according  to  the  Hofmeister 
classification,  is  based  on  a  misconception  of  the 
application  of  ammonium  sulphate  in  separating 
proteids  by  their  precipitation  characters.  While 
there  seem  to  be  some  differences  in  the  dis- 
tribution of  the  antibodies  in  individual  specific 
sera  in  comparative  experiments,  this  is  not  so 
absolute  as  maintained  by  Pick  *  and  others.  Gib- 
son's work  on  the  fractionating  of  poly  agglutina- 
tive serum  shows  that  no  separation  of  the  several 
antibodies  developed  in  an  individual  serum  is 
possible.  In  the  case  of  antitoxic  sera  both  Gibson 
and  Ledingham  find  that  in  goat  serum  the  antitoxin 
is  not  invariably  associated  with  the  euglobulin 
fraction  as  maintained  by  Pick,  but  shows  the  same 
solubilities  as  that  in  horse  serum. 

Toxins  and  other  Poisonous  Cell  Derivatives,  in 
General.  — •  Soon  after  bacteriology  had  demon- 
strated the  etiological  connection  between  bacteria 
and  disease,  the  conviction  gained  ground  that  it 

1  Pick,  Beitrage  z.  chera.  Physiol.  u.  Pathol.,  Vol.  i,  1901. 


20  IMMUNE  SERA 

was  less  the  actual  destruction  wrought  by  the 
bacteria  directly,  than  the  injury  produced  by  their 
chemical  products  that  gave  rise  to  the  lesions  in 
the  infectious  diseases.  Brieger,  especially,  was 
one  of  the  first  to  direct  attention  to  the  probable 
existence  of  specific  poisons  in  the  bacteria.  He 
isolated  a  number  of  well  defined  chemical  sub- 
stances called  ptomaines,  most  of  which  were  highly 
toxic.  Subsequent  study,  however,  showed  that 
these  were  not  the  specific  bacterial  poisons.  The 
latter,  the  true  toxins  are  something  quite  different 
as  we  shall  see  in  a  moment.  Still  later  other 
substances  were  isolated  from  bacteria,  and  these 
were  termed  toxalbumins.  We  now  know  that 
some  of  these  were  identical  with  the  true  toxins, 
but  that  others  were  entirely  unrelated. 

What  then  are  the  true  toxins?  A  number  of 
pathogenic  bacteria,  when  grown  in  pure  culture, 
produce  dissolved  poisons  in  the  culture  fluid. 
These  poisons  are  neither  ptomaines  nor  proteid 
substances;  their  chemical  nature  is  still  absolutely 
unknown.  They  are  extremely  sensitive  to  exter- 
nal influences,  especially  against  heat,  and  in  many 
ways  are  very  analogous  to  ferments.  Physio- 
logically the  toxins  are  extremely  poisonous,  far 
beyond  that  of  any  of  the  ordinary  well  known 
poisons,  and  this  poisonous  action  manifests  itself 
only  after  a  certain  latent  period  known  as  the 
period  of  incubation.  Finally  one  of  the  funda- 


A  NTITOXINS  2 1 

mental  properties  of  the  toxins  is  their  ability 
to  excite,  in  the  organism  attacked,  antitoxins 
directed  specifically  against  them,  so  that  for  every 
true  toxin  there  is  a  corresponding  antitoxin. 

In  addition  to  these  bacterial  toxins  we  know 
of  other  poisonous  substances  possessing  similar 
characteristics.  Among  these  are  the  "  zootoxins," 
-  snake  venoms,  spider  and  toad  poisons,  the 
toxin  of  eel  blood,  and  the  "  phytotoxins," 
ricin,  crotin,  abrin,  etc.  It  may  be  mentioned  that 
some  of  these  are  of  somewhat  more  complex  con- 
stitution than  the  ordinary  bacterial  toxins.  Ricin, 
for  example,  appear  to  possess  one  haptophore 
group  but  two  ergophore  groups,  a  toxic  and  an 
agglutinating  one.  In  the  case  of  the  snake 
venoms  it  is  not  yet  definitely  known  whether  they 
are  haptins  of  the  first  order  or  of  the  second. 

The  Relations  Existing  between  Toxin  and  Anti- 
toxin. —  The  exact  nature  of  the  toxin-antitoxin 
reaction  has  long  been  the  subject  of  study  and  has 
given  rise  to  considerable  discussion.  For  obvious 
reasons  most  of  the  work  has  been  done  with 
diphtheria  and  tetanus  toxins  and  their  antitoxins. 
In  order  to  give  the  reader  some  conception  of 
the  diverging  views  of  various  authorities  we  shall 
devote  a  few  pages  to  a  brief  study  of  the  diphtheria 
toxin-antitoxin  reaction. 

During  the  earlier  years  of  toxin-antitoxin  in- 
vestigations the  filtered  or  sterilized  bouillon,  in 


22  IMMUNE   SERA 

which  the  diphtheria  bacillus  had  grown  and  pro- 
duced its  "  toxin,"  was  supposed  to  require  for 
its  neutralization  an  amount  of  antitoxin  directly 
proportional  to  its  toxicity  as  tested  in  guinea  pigs. 
Thus,  if  from  one  bouillon  culture  ten  fatal  doses 
of  "toxin"  were  required  to  neutralize  a  certain 
quantity  of  antitoxin,  it  was  believed  that  ten 
fatal  doses  from  every  culture,  without  regard  to 
the  way  in  which  it  had  been  produced  or  preserved, 
would  also  neutralize  the  same  amount  of  antitoxin. 
Upon  this  belief  was  founded  the  Behring-Ehrlich 
definition  of  an  antitoxin  unit.1 

The  results  of  tests  by  different  experimenters 
of  the  same  antitoxic  serum,  but  with  different  diph- 
theria toxins,  proved  this  opinion  to  be  incorrect. 
Ehrlich2  deserves  the  credit  for  first  clearly  per- 
ceiving and  calling  attention  to  this  fact.  He 
obtained  from  various  sources  twelve  toxins  and 
compared  their  neutralizing  value  upon  antitoxin; 
these  tests  gave  interesting  and  important  in- 
formation. The  following  table  gives  the  results 
in  four  of  his  toxins  and  well  illustrates  the  point  in 
question : 

1  This  unit  was  "  ten  times  the  amount  of  antitoxic  serum 
necessary  to  just   protect   a    250   gramme  guinea   pig   against 
ten  fatal  doses  of  the  toxin." 

2  Ehrlich,    Die    Werthbemessung    des    Diphtherieheilserums., 
Klinisches  Jahrbuch,    1897. 


ANTITOXINS 


Smallest  num- 

Fatal doses  re- 

ber of  fatal 

quired  to 

doses  of  toxic 

"  completely 

Estimated 

bouillon  re- 

neutralize "  one 

L+  minus 

Serial 

minimal  fatal 

quired  to  kill  a 

antitoxin  unit 

Num- 
her. 

dose  for 
250  gm. 
guinea  pigs. 

250  gm.  guinea 
pig  within 
5  d'iys  when 

as  determined 
by   the  health 
of  the  guinea 

LO  in 
fatal 
doses. 

Remarks. 

mixed  with  one 

pi-?  re-naining 

nntitoxin  unit. 

una'Tectcd. 

("  Lt  Ehriich.  ') 

("  LO  Ehriich.") 

A 

o  .  009  cc. 

39-4 

33-4 

6 

Old;        deterio- 

rated from  o.oo  3 

to  o  .  009. 

B 

0.0165  cc 

76.3 

54-4 

1   "> 

Fresh          toxin, 

preserved     with 

tricresol. 

C 

o  .  039  cc. 

123. 

108. 

15 

A     number      of 

fresh      cultures, 

grown  at  37°  C. 

four    and    eight 

days. 

D 

o  .  0025  cc. 

100 

5° 

5° 

Tested  immedi- 

ately   after     its 

withdrawal. 

It  was  natural  to  suppose,  as  the  early  investi- 
gators did,  that  a  just  neutral  mixture  of  toxin  and 
antitoxin,  would  require  the  addition  of  but  one 
fatal  dose  of  toxin  in  order  to  regularly  kill  the  test 
animal.  In  the  above  table,  however,  we  see  that 
this  difference  ranges  from  six  to  fifty  fatal  doses. 

Partial  Saturation  Method  -  -  Toxons,  Toxoids.  - 
Ehriich  obtained  considerable  additional  informa- 
tion by  means  of  his  "  partial  saturation  "  method. 
Certain  experiments  had  led  him  to  believe  that  the 
original  antitoxin  on  which  he  had  based  his  "  unit  " 
determinations,  while  able  to  neutralize  100  fatal 
doses  (per  unit)  really  represented  200  "  binding 


IMMUNE   SERA 


units,"  and  that  the  toxic  bouillon  really  contained 
several  kinds  of  poisonous  substances  able  to  com- 
bine with  antitoxin. 

He  now  believes  that  the  diphtheria  bacilli  excrete 
at  least  two  such  poisons,  "  toxins  "  and  "  toxons ;  " 
that  these  very  quickly  decompose  to  a  greater  or 
less  extent  forming  various  "  toxoids." 

In  the  case  of  a  hypothetically  pure  toxin  Ehrlich 
believes  that  one  antitoxic  unit  would  correspond 
to  200  fatal  doses  or  200  binding  units.  If  the 
entire  amount  of  antitoxin,  i.e.  !§§  is  added  to 
the  amount  of  toxin  in  question,  the  result  will  be 
just  complete  neutralization.  If  the  toxin  is  entirely 
pure,  JJ§  of  the  antitoxin  unit  would  neutralize  all 
but  ?A(j  of  the  initial  toxicity  and  M§,  or  £{$  or  /A, 
etc.  of  the  antitoxin  added  would  permit  correspond- 
ing degrees  of  toxicity  to  be  demonstrated  through 
animal  inoculations.  It  was  found,  however,  that 
neutralization  according  to  this  simple  scale  did  not 
take  place.  The  results  were  complicated  and  Ehrlich 


Toxon 


0     10   20    30    40    50    60    70    80   90  100 

FIG.  2. 


150 


200 


found  it  convenient  to  express  them  graphically  in 
the  form  of  the  so-called  "  toxin  spectra."     Without 


ANTITOXINS  25 

going  much  deeper  into  the  subject  the  point  may  be 
illustrated  by  the  appended  diagrams  or  "  spectra." 

Fig.  2  shows  the  simplest  conceivable  diphtheria 
poison.  In  this  case  the  following  values  would 
be  obtained. 

xcc  poison  (100  fatal  doses)  +  f§£  antitoxin 
units  =  o,  i.e.  absolutely  neutral. 

xcc  poison  +  M§  --  =  Free  toxon. 

xcc  poison  +  iU  --=  Free  toxon. 

That  is  to  say,  if  the  proportion  of  antitoxin  added 
was  £{}§  of  the  amount  required  for  complete 
neutralization,  it  would  be  found  that  the  poison 
thus  uncombined  was  much  less,  and  differently 
toxic  than  a  corresponding  amount  of  the  original 
toxin.  It  was  found  that  these  fractions  possessed 
a  rather  constant  though  low  degree  of  toxicity 
with  characteristic  action.  This  consisted  in  the 
production  of  some  local  oedema,  followed  by  a 
long  incubation  period,  and  finally  the  develop- 
ment of  cachexia  and  paralysis.  Ehrlich  believes 
that  this  action  is  due  to  a  separate  poison  excreted 
by  the  diphtheria  bacillus  which  he  calls  a  toxon. 

If  we  continue  with  the  above  poison  we  shall 
obtain  these  values: 

xcc  poison  +  sV<r  =  Toxin  action    (i  fatal  dose). 

xcc  poison  +  /A  =30  fatal  doses. 

x™  poison  +  WV  =  90  fatal  doses,  etc. 

That  is  to  say,  if  we  add  only  &9ff  units  antitoxin, 
i.e.  sJfli  unit  less  than  in  the  J&J  mixture,  we  find 


26 


IMMUNE  SERA 


that  one  fatal  dose  is  set  free.  This  relation  would 
exist  right  to  the  end.  The  fact  that  in  this  experi- 
ment the  toxins  are  liberated  after  the  toxons, 
shows  that  the  toxons  have  less  affinity  for  the  anti- 
toxin than  have  the  toxins. 

As  a  matter  of  fact,  however,  conditions  are  prob- 
ably never  as  simple  as  this.  In  the  process  of 
toxin  formation  a  double  action  is  always  going 
on  —  that  of  toxin  and  toxon  production,  and  that 
of  their  decomposition.  As  was  pointed  out  on 
a  previous  page  the  poisons  quickly  change  into 
non-poisonous  toxoids,  and  these  substances  are 
still  able  to  bind  antitoxin. 

This  is  shown  in  the  following  "  spectrum." 


Protoxoid 


0    10   20   30   40    50 


100 


Toxon 


150  160 


FIG.  3. 


Here  we  would  obtain  the  following  figures: 

xcc  poison  +  f$$  antitoxin     unit  =  o,   i.e.     abso- 
lutely neutral. 

xcc  poison  +  i{j-<|-  =  Toxon  free. 

xcc  poison  +  Mo  =  =  Toxon  free. 

XCG  poison  +  Mf  ----  Toxin  free  (i  fatal  dose.) 

xcc  poison  +  iH  =  Toxin  free  (60  fatal  doses.  ) 

xcc  poison  +  5eo\  =  Toxin  free  (100  fatal  doses.) 


ANTITOXINS 


Now  we  come  to  the  non-poisonous  "prototoxoids"  : 
xcc  +  -/^  ==  Toxin  free(ioo  fatal  doses.) 
^cc  +  ^  =  Toxin  free  (100  fatal  doses.) 
xcc  +  3^0   =  Toxin  free  (100  fatal  doses.) 
We    see   here    that    after   we   have   reduced    the 
antitoxin   to  /o°o  no  further   increase   of   toxicity  is 
brought  about  by  any  further  reductions.     Ehrlich 
calls   these   toxoids    "  prototoxoids  "   because   they 
have  such  a  high  affinity  for  the  antitoxin.     But 
there  are  apparently  still  other  toxoids,  as  is  shown 
by  the  following  spectrum : 


Protoxoid 


Syntoxoid 


Toxon 


100  10  200 

FIG.  4. 

Here  we  would  obtain  values  as  follows: 
xec  poison  +  f  |j{y  ==  o,  i.e.  absolutely  neutral. 
xcc  poison  +  M<T  =  Toxon. 
xcc  poison  +  Mf  =  Toxin  free  (i  fatal  dose). 
xcc  poison  +  M|  --=  Toxin  free  (  2  fatal  doses.) 
xcc  poison  +  M§  = :  Toxon  free  (30  fatal  doses.) 
Here   we   find   that   in   the   middle   part   of   the 
"  spectrum  "  we  encounter  a  zone  in  which  each  •%%-$ 
antitoxin  unit  neutralizes  one  fatal  dose.     Ehrlich 
believes  that  this  part  of  the  mixture  consists  of 


28  IMMUNE  SERA 

equal  parts  of  syntoxoid  and  toxin  —  that  is  to 
say,  he  believes  there  are  also  toxoids  which  have 
the  same  degree  of  affinity  for  antitoxin  that  this 
toxic  has.  He  speaks  of  these  as  "  syntoxoids." 

Views  of  Arrhenius,  Bordet  and  Others.  — -  Bordet 
and  others  refuse  to  accept  Ehrlich's  views  and 
the  whole  matter  is  at  the  present  time  under 
active  discussion.  Thus  the  existence  or  non- 
existence  of  toxons  has  excited  a  great  deal  of  dis- 
cussion among  investigators.  The  great  Swedish 
chemist,  Arrhenius,  has  recently  given  much  atten- 
tion to  the  toxins  and  has  applied  the  principles  of 
physical  chemistry  to  the  toxin-antitoxin  reaction. 
It  is,  of  course,  well  known  that  a  solution  of  a  com- 
pound such  as  sodium  chloride  represents  not  only 
NaCl  in  solution,  but  also  sodium  ions  and  chlorine 
ions.  There  is  a  certain  amount  of  dissociation 
going  on  hand  in  hand  with  a  combination  of  the 
two  components.  The  degree  of  this  varies  with 
the  temperature  and  the  dilution  of  the  substances. 
Arrhenius  believes  that  the  same  process  goes  on 
with  the  toxin-antitoxin  combination  and  that 
such  more  or  less  dissociated  compounds  give  rise 
to  the  effects  Ehrlich  ascribes  to  the  toxons. 

Bordet  has  attempted  to  explain  the  toxon 
phenomena  in  a  different  way.  He  shows  that  the 
toxin  molecule  can  combine  with  antitoxin  in 
varying  proportions.  One  would  then  assume  that 
the  toxin  molecule  possesses  several  "  binding  " 


ANTITOXINS  29 

groups.  The  complete  occupation  of  these  groups 
causes  the  toxicity  to  be  entirely  lost,  whereas 
partial  saturation  so  affects  the  molecule  that  it 
exerts  a  milder  and  different  action. 

The  principles  of  colloid  chemistry  have  also 
been  applied  to  the  study  of  the  toxin-antitoxin 
combination.  Field1  has  recently  tested  the  electri- 
cal charge  of  toxins  and  antitoxins  and  finds  that 
both  diphtheria  and  tetanus  toxin  and  their  anti- 
toxins are  electropositive,  passing  to  the  cathode 
pole.  He  concludes  that  the  combination  of  toxin 
and  antitoxin  may  perhaps  represent  not  a  true 
chemical  reaction,  but  the  absorption  of  one  colloid 
by  another.  Ehrlich,  however,  still  adheres  to  his 
views  and  points  out  that  the  advocates  of  colloid 
chemistry  have  been  compelled  to  assume  the 
existence  of  specific  atomic  groupings  very  much 
after  his  own  ideas.  He  also  cites  van  Calcar2  who 
claims  to  have  separated  toxin  and  toxon  by  a 
dialyzing  procedure. 

1  Field  and  Teague,  Journ.  of  Exper.  Medicine,  Vol.  ix,  1907. 

2  van  Calcar,  Berlin,  Klin  Wochenschr,  1905. 


AGGLUTININS 

The  Agglutination  Phenomenon.  —  We  have  just 
seen  that  pathogenic  bacteria  may  be  divided  into 
those  which  produce  extracellular  toxins  in  culture 
media,  and  those  which  do  not.  Against  the 
former  the  organism  defends  itself  by  the  production 
of  antitoxins ;  against  the  latter  it  produces  a  variety 
of  antibodies :  —  bacteriolysins,  agglutinins,  precipi- 
tins,  opsonins  and  possibly  others. 

The  agglutinins  can  be  observed  either  in  a  test- 
tube  or  in  a  microscopical  preparation.  For  example, 
if  typhoid  or  cholera  immune  sera  are  added  respec- 
tively to  a  24-hour  culture  of  typhoid  or  cholera 
bacilli,  and  the  mixture  placed  in  a  thermostat, 
the  following  phenomenon  will  be  noticed:  The 
bacteria  which  previously  clouded  the  bouillon 
uniformly,  clump  together  into  little  masses,  settle 
to  the  sides  of  the  test-tube  and  gradually  fall  to 
the  bottom  until  the  fluid  is  almost  entirely  clear. 
In  a  control  test,  on  the  contrary,  to  which  no  active 
serum  is  added,  the  fluid  remains  uniformly  cloudy. 
The  reaction  is  completed  in  twenty-four  hours 
at  the  most.  If  the  reaction  is  observed  in  a  hang- 
ing drop,  it  is  seen  that  the  addition  of  the  active 
serum  first  produces  an  increased  motility  of  the 

3° 


AGGLUTININS  31 

bacteria  which  lasts  a  short  time  and  is  followed 
by  a  gradual  formation  of  clumps.  One  gets  the 
impression  that  the  bacteria  are  dying  together. 
Frequently  one  sees  bacteria  which  have  recently 
joined  a  group  make  violent  motions  as  though  they 
were  attempting  to  tear  themselves  away;  then 
they  gradually  lose  their  motility  completely.  Even 
the  larger  groups  of  bacteria  may  exhibit  movement 
as  a  whole.  After  not  more  than  one  or  two  hours 
the  reaction  is  completed;  in  place  of  the  bacteria 
moving  quickly  across  the  field,  one  sees  one  or 
several  groups  of  absolutely  immobile  bacilli.  Now 
and  then  in  a  number  of  preparations  one  sees 
a  few  separate  bacteria  still  moving  about  among 
the  groups.  If  the  reaction  is  feeble,  either  because 
the  immune  serum  has  been  strongly  diluted  or 
because  it  contains  very  little  agglutinin,  the  groups 
are  small  and  one  finds  comparatively  many  iso- 
lated and  perhaps  also  moving  bacteria.  It  is 
essential  each  time  to  make  a  control  test  of  the 
same  bacterial  culture  without  the  addition  of 
serum.  Under  some  circumstances  the  reaction 
proceeds  with  extraordinary  rapidity  so  that  the 
bacilli  are  clumped  almost  immediately.  By  the 
time  the  microscopical  slide  has  been  prepared 
and  brought  into  view  nothing  is  to  be  seen 
of  any  moving  or  isolated  bacteria,  and  only  by 
means  of  the  control  test  is  it  possible  to  tell 
whether  the  culture  possessed  normal  motility. 


32  IMMUNE  SERA 

We  are  not  yet  informed  as  to  the  nature  of  these 
phenomena.  A  number  of  theories  have  been  ad- 
vanced, into  which,  however,  we  cannot  here  enter. 

In  some  cases  the  agglutinins  are  active  even  in 
very  high  dilutions.  Thus  in  typhoid  patients 
and  typhoid  convalescents  a  distinct  agglutination 
has  been  observed  in  dilutions  of  i :  5000,  and  this 
action  persisted  for  years,  though  not,  of  course, 
in  the  same  degree.  Even  normal  blood-serum, 
when  undiluted,  often  produces  agglutination.  But 
the  above  specific  agglutinins,  which  do  not  exist 
beforehand,  being  formed  only  in  consequence  of 
an  infection,  are  characterized  by  this,  that  the 
agglutination  occurs  even  when  the  serum  is  diluted 
(at  least  i  :  30  to  i  .'50),  and,  furthermore,  that  after 
this  dilution  the  action  is  still  specific,  i.e.  cholera 
immune  serum  agglutinates  only  cholera  bacilli, 
typhoid  immune  serum  only  typhoid  bacilli,  etc. 
This  specificity,  however,  as  will  be  shown  later, 
is  not  always  absolute. 

Agglutinins  can  also  be  developed  against  red 
blood  cells  and  against  certain  protozoa  (trypan- 
osomes).  We  speak  of  the  former  as  hamag- 
glutinins.  Analogous  to  the  haemolytic  action  or 
normal  serum  on  the  red  cells  of  certain  other 
species,  we  find  that  normal  serum  is  able  to 
agglutinate  the  red  cells  of  many  species  and  bac- 
teria. For  example,  normal  goat  serum  aggluti- 
nates the  red  cells  of  man,  pigeon,  and  rabbit; 


AGGLUTININS  33 

normal  rabbit  serum  agglutinates  typhoid  and 
cholera  bacilli. 

Purpose  of  Agglutination.  —  It  is  not  yet  clear 
what  the  purpose,  if  any,  of  the  agglutinating 
function  is.  Gruber,  the  first  to  thoroughly  study 
and  appreciate  the  bacterial  agglutinins,  assumes 
that  the  process  injures  the  affected  cell,  preparing 
it  for  solution  and  destruction.  After  numerous 
experiments  I  have  not  been  able  to  convince 
myself  of  any  damaging  influence  of  the  agglutinins 
on  the  affected  cell,  be  this  blood  cell  or  bacterium, 
and  the  observations  of  other  authors  confirm  this 
opinion.  Agglutinated  bacteria  are  capable  of 
living  and  of  reproduction,  and  agglutinated  red 
blood  cells  are  no  more  fragile  or  easier  to  destroy 
than  normal,  non-agglutinated  cells.  Neither  can 
anything  be  discovered  microscopically  which  would 
indicate  any  injury  to  their  structure. 

One  thing  is  certain:  that  the  agglutinins  are  in 
no  way  related  to  the  lysins  found  in  serum,  and 
so  of  course  are  not  identical  with  these.  The 
simultaneous  occurrence  in  a  serum  of  immune 
bodies,  interbodies,  complements,  and  agglutinins 
is  an  entirely  independent  phenomenon  which  is 
in  no  way  regular.  There  are  sera  which  dis- 
solve certain  cells  without  agglutinating  them,  and 
others  which  agglutinate  cells  without  dissolving 
them. 

Historical.  —  Serum   diagnosis  by  means   of  the 


34  IMMUNE   SERA 

agglutinins  was  introduced  chiefly  through  the 
labors  of  Gruber  and  Widal.  The  studies  under- 
taken by  Gruber  and  his  pupil  Durham  began  as 
early  .as  1894.  At  the  Congress  for  Internal  Medi- 
cine in  1896  l  Gruber  first  announced  that  he  had 
discovered  the  reaction  in  typhoid  convalescents, 
and  asked  that  his  observations  be  verified  if  pos- 
sible. Soon  after  this  Pfeiffer  and  his  co-workers 
published  a  study  which  confirmed  Gruber 's  results.2 
The  significance  of  the  reaction  as  a  diagnostic 
help  was  unquestionably  first  pointed  out  by  Widal,3 
who  showed  that  the  reaction  appears  at  a  relatively 
early  period  of  the  disease,  and  may  therefore  be 
employed  as  a  diagnostic  measure.  We  must  not 
omit  to  state  that  Griinbaum4  in  March,  1896,  several 
months  before  Widal's  publication,  had  also  grasped 
the  significance  of  the  reaction  as  a  diagnostic 
measure.  Owing  to  insufficient  clinical  material 
his  publication  did  not  appear  until  some  time  after 
Widal's.  Hence,  in  acknowledgment  of  the  labors 
of  the  two  authors  most  concerned  in  the  discovery 
and  introduction  of  this  reaction,  we  now  speak 
of  it  as  the  "  Gruber- Widal  reaction,"  whereas  in 

1  Transactions  of  the  Congress,  edited  by  E.  von  Leyden  and 
R.  Pfeiffer,  Wiesbaden,  1896. 

2  Pfeiffer  and   Kolle,  Deutsche    med.    Wochenschrift,   1896, 
No.  12. 

3  Widal,  Bulletin  de  la  soc.  me'd.  des  hop.,  June  26,  1896. 

4  Grunbaum,  Lancet,  Sept.  19,  1896;  Muench.  med.  Wochen- 
schrift, 1897,  No.   13;  Blood  and  the  identification  of  bacterial 
species,  Science  Progress,  Vol.  I,  No.  5,  1897. 


AGGLUTININS  35 

the  beginning  only  the  term  '  *  Widal  reaction  ' ' 
was  used. 

The  manner  in  which  the  reaction  proceeds  in 
microscopical  preparations  as  well  as  when  mac- 
roscopically  observed  has  been  described  above 
(page  30).  Nowadays  the  microscopic  method  is 
given  the  preference  l  because  in  many  cases  it  is 
distinct  when  the  macroscopic  reaction  fails;  and 
further  because  the  former  yields  distinct  results 
within  an  hour  at  the  most,  whereas  in  many  cases 
twenty-four  hours  are  required  for  the  macroscopic 
test. 

Pfaundler's  Reaction  (Thread  Reaction).  —  It 
may  be  well  at  this  point  to  call  attention  to  a 
peculiar  reaction  described  by  Pfaundler  2  in  1896. 
This  author  showed  that  certain  bacteria,  though 
they  might  not  be  agglutinated  by  a  given  serum, 
would  often,  when  they  were  grown  therein,  develop 
in  the  form  of  long  threads  more  or  less  interlaced. 
This  occurred  only  in  the  specific  serum  and  was 
absent  in  the  normal  serum.  Most  authorities 
regard  the  thread  reaction  as  a  manifestation  of 
agglutinins.  According  to  Metchnikoff  this  reaction 
sometimes  gives  more  information  concerning  a 
,serum  than  does  the  ordinary  agglutination  test. 

Nature  of  the  Agglutinins.  -  -  The  agglutinins  are 

1  This  applies  to  typhoid ;  in  other  diseases  the  macroscopic 
method  is  sometimes  preferable, 

2  Pfaundler,  Centralblatt  Bacteriologie,  Vol.  xix,  1896. 


36  IMMUNE  SERA 

fairly  resistant  substances  which  withstand  heat- 
ing to  60°  0.,  and  lose  their  power  only  on  heating 
to  65°  C.  It  is  possible,  therefore,  to  make  a  serum 
bacteriolytically  inactive  by  heating  to  55°C.,  and 
still  preserve  its  agglutinating  power.  Corres- 
ponding to  the  specific  combining  power  of  these 
agglutinins,  they  possess  a  haptophore  group  which 
effects  the  combination,  and  a  second  group,  easily 
decomposed  by  acids,  which  effects  the  clumping. 
In  the  bacterium  as  well  as  in  the  blood  cell  there 
exists  a  substance  not  yet  closely  studied,  called 
the  agglutinable  substance.  This  also  has  two  groups, 
a  haptophore,  which  combines  with  the  hapto- 
phore group  of  the  agglutinin;  and  a  second,  more 
delicate  group,  which  is  acted  on  by  the  functional 
group  of  the  agglutinin. 

Nature  of  the  Agglutination  Reaction.  —  The  union 
of  agglutinin  with  the  agglutinable  substance  is  a 
chemical  reaction,  and  is  quantitative.  The  amount 
of  bacteria  in  the  emulsion  used  to  test  the  amount 
of  agglutinin  must,  therefore,  be  known.  An 
emulsion  one  hundred  times  as  dense  as  another 
would  require  one  hundred  times  as  much  agglu- 
tinin to  give  an  equally  complete  reaction.  Agglu- 
tinin acts  both  on  living  and  on  dead  bacteria. 

The  influence  of  salts  upon  agglutination  is  in 
a  sense  comparable  to  their  action  upon  the  pre- 
cipitins.  Joos  found  that  antityphoid  serum  did 
not  agglutinate  typhoid  bacilli  in  the  absence  of 


w,  AGGLUT1N1NS  37 

'  ».  c 

salts.  For  agglutination  to  take  place  he  con- 
siders  it  is  as  necessary  as  the  agglutinin  and  agglu- 
tinable  substance.  He  believes  that  salts  play  an 
active  part  in  the  process,  a  conception  which  is 
contrary  to  Bordet's,  that  the  absence  of  salts 
offers  only  a  physical  impediment  to  agglutination. 
Friedberger  does  not  consider  that  the  salts  act 
chemically  for  he  found  agglutination  to  take  place 
in  the  presence  of  grape  sugar,  asparagin,  etc. 

In  view  of  the  fact  that  the  protoplasm  of  the 
body  and  the  albuminous  constituents  of  serum  have 
a  close  relationship  to,  or  really  are,  colloids,  inves- 
tigators have  studied  certain  reactions  which  occur 
among  the  colloids  with  the  expectation  that  these 
would  throw  some  light  on  the  reactions  of  proto- 
plasm and  of  serums, 

Colloids  diffuse  very  slowly  and  exert  little  or  no 
osmotic  pressure,  supposedly  because  of  the  large 
size  of  the  particles.  They  do  not  conduct  elec- 
tricity, but  the  particles  react  to  the  electric  current 
by  alterations  in  the  direction  of  their  motion  (i.e. 
toward  the  positive  or  the  negative  pole),  and, 
moreover  carry  electric  charges  themselves. 

The  features  of  colloids  which  bring  them  into 
relation  with  the  subject  in  hand  are  their  coagul- 
able  nature  in  certain  instances  and  the  fact  that 
their  particles  may  be  agglutinated  or  precipi- 
tated by  the  addition  of  minute  amounts  of  salts 
(electrolytes).  This  of  course  is  entirely  analogous 


38  IMMUNE  SERA 

to  the  need  of  salts  in  the  agglutination  of  bacteria 
by  sera.  In  the  latter  reaction  the  agglutinins 
carry  a  positive,  the  bacteria  a  negative  charge. 
The  resulting  combination,  therefore,  does  not 
precipitate  from  the  menstruum,  supposedly  because 
there  is  still  sufficient  difference  in  the  electric 
potential.  When  salts  are  present  the  kations  so 
alter  the  electric  conditions  of  the  colloidal  par- 
ticles, i.e.,  of  the  agglutinin-bacterium  combina- 
tion, that  their  surface  tension  is  increased.  In 
order  to  overcome  this  the  particles  get  together, 
presenting  in  a  clump  less  surface  tension  than  if 
they  remained  as  individual  particles. 

Agglutinoids.  —  Agglutinins  which  have  lost  their 
agglutinophore  group  through  the  action  of  acids, 
etc.,  but  which  still  possess  their  haptophore  group, 
are  called  agglutinoids,  just  as  toxins  which  have 
lost  their  toxophore  group  are  called  toxoids. 
Such  agglutinoids,  then,  may  still  combine  with 
the  blood  cells  or  bacteria  without  being  able,  how- 
ever, to  produce  any  clumping  or  agglutination. 
The  nature  of  agglutinoids,  however,  is  still  very 
obscure  as  is  also  the  means  by  which  they  inhibit 
agglutination.  It  has  occasionally  been  observed, 
for  example,  that  agglutination  is  absent  in  con- 
centrated serum,  and  present  in  dilute  serum. 
This  zone,  of  no  agglutination  preceding  that  of 
agglutination  is  often  spoken  of  as  the  pro  zone. 
It  has  been  explained  as  due  to  the  presence  in  the 


AGGLUTININS  39 

serum  of  agglutinoids.  These  are  assumed  to 
possess  a  higher  affinity  for  the  bacteria  than  do 
the  agglutinins  and  so  prevent  the  latter  from 
acting  on  the  bacteria.  Since,  however,  the  agglu- 
tinins are  usually  far  more  abundant  than  the 
agglutinoids,  dilution  of  the  serum  dilutes  the  latter 
to  practically  nothing,  thus  allowing  the  agglutinins, 
to  combine  with  the  bacteria.  Some  recent  experi- 
ments by  Field  show  that  the  pro  zone*  may  have 
an  entirely  different  explanation,  based  on  behavior 
of  bacteria  and  agglutinin  as  colloids.  It  has 
already  been  stated  that  the  union  of  agglutinin 
and  bacterium  does  not  precipitate  because  there 
is  still  sufficient  electric  potential ;  the  combination 
carries  a  negative  charge.  Field  believes  that  with 
very  large  amounts  of  agglutinin  (as  in  the  pro 
zone]  the  bacteria  load  themselves  with  so  much 
agglutinin  that  the  combination  now  carries  a 
considerable  positive  charge.  The  surface  tension 
therefore  is  not  sufficient  to  cause  a  clumping  to 
occur.  Naturally  the  presence  of  salts  does  not 
alter  the  condition  as  the  kations  also  carry  a 
positive  charge. 

Group  Agglutinins.  —  For  some  time  after  their 
discovery  the  agglutinins  were  regarded  as  strictly 
specific,  i.e.  a  serum  derived,  for  example,  from  a 
typhoid  infection  would  agglutinate  only  typhoid 
bacilli  and  no  others.  After  a  time,  however,  it  was 
found  that  such  a  serum  would  frequently  aggluti- 


40  IMMUNE  SERA 

nate  somewhat  related  organisms,  though  not, 
usually,  to  so  high  a  degree.  In  other  words,  while 
agglutinins  may  be  nearly,  if  not  quite,  specific  in 
their  action,  a  serum  which  produces  agglutination 
may  be  far  from  being  so. 

The  following  examples  will  illustrate  the  point. 
In  a  case  of  infection  with  paratyphoid  bacilli, 
type  B,  the  bacilli  of  the  infecting  type  B  were 
agglutinated  1:5700;  typhoid  bacilli,  however,  only 
1:120,  while  paratyphoid  bacilli  type  A  were  not 
agglutinated  at  all.  In  a  case  of  typhoid  infection 
an  agglutination  with  a  dilution  of  i :  40  was  obtained 
for  paratyphoid  type  B,  while  typhoid  bacilli  were 
agglutinated  in  a  dilution  of  i :  300  and  over.  As  a 
rule  the  agglutination  with  the  infecting  agent  is  by 
far  the  strongest,  i.e.  it  proceeds  even  in  high  dilu- 
tions, whereas  other  bacteria  require  a  stronger 
concentration. 

In  all  this  we  are  dealing  with  the  same  phenom- 
enon which  undoubtedly  plays  a  role  in  the  agglu- 
tination with  blood  of  icteric  patients,  the  so-called 
group  agglutination,  as  it  was  first  termed  by  Mein- 
hard  Pfaundler.1  The  bacteria  which  are  aggluti- 
nated by  one  and  the  same  serum  need  not  at  all 
be  related  in  their  morphological  or  other  biolog- 
ical characteristics,  as  Pfaundler  at  first  assumed. 
Conversely,  micro-organisms  which,  because  of  the 

1  tiber  Gruppenagglutination  und  das  Verhalten  des  Bacte- 
rium coli  bei  Typhus,  Muench.  mcd.  Wochenschrift,  1899,  No.  15. 


AGGLUTININS  4! 

characteristics  mentioned,  are  regarded  as  entirely 
identical  or  almost  so,  are  sharply  differentiated  by 
means  of  their  agglutination.  In  other  words,  the 
"  groups,"  arrived  at  by  means  of  a  common  agglu- 
tination sometimes  have  no  relation  to  species  as  the 
term  is  usually  employed.  Thus,  according  to  Stern, 
certain  varieties  of  proteus  and  of  staphylococci  ex- 
cite the  production  of  sera  which  exert  marked  agglu- 
tinating powers  also  on  typhoid  bacilli,  although 
otherwise  we  do  not  regard  these  three  micro- 
organisms as  at  all  related.  On  the  other  hand  by 
means  of  agglutination  we  can  sharply  distinguish 
cholera  bacilli  from  their  nearest  related  species. 
Because  of  this  lack  of  absolute  specificity  the 
serum  diagnosis  of  infection  or  the  identification  of 
bacteria  has  value  only  when  very  carefully  tested. 
Absorption  Methods  for  Differentiating  between 
a  Mixed  and  a  Single  Injection.  —  In  1902,  Castellani 1 
called  attention  to  a  procedure  which  consists  in 
saturating  the  diluted  immune  serum  with  succes- 
sive quantities  of  the  bacteria  most  strongly  agglu- 
tinated until  the  agglutinating  power  for  these 
bacteria  =  o.  After  centrifuging  the  mixture  the 
clear  fluid  is  tested  on  the  second  variety  of  bacteria, 
and  from  this  one  learns  whether  mixed  or  single 
infection  was  present.  According  to  Castellani  if 
the  serum  of  an  animal  immunized  against  a  certain 
microorganism  is  saturated  with  that  organism, 

1  Castellani,  Zeitschrift  Hygiene,  Vol.  xl,  1902. 


IMMUNE  SERA 


the  serum  will  lose  its  agglutinating  power  not  only 
for  that  organism  but  also  for  all  other  varieties  that 
it  formerly  acted  on.  Saturated  with  the  others,  its 
action  upon  the  first  is  reduced  little  or  not  at  all. 

The  serum  of  an  animal  immunized  against  two 
microorganisms  A  and  B,  loses  its  agglutination 
when  saturated  with  A,  only  for  A.  Saturated 
with  A  and  B,  it  loses  agglutinating  power  for  both. 

Park,1  who  has  devoted  considerable  attention  to 
this  subject  finds  that  the  absorption  method 
simply  proves  that  when  one  variety  of  bacteria 
removes  all  agglutinins  for  a  second,  the  agglu- 
tinins  under  question  were  not  produced  by  that 
second  variety. 

Specific  and  group  agglutinins  may  perhaps  be 
better  understood  by  means  of  the  following  dia- 
gram. We  assume  that  the  typhoid  bacillus  pos- 


B        C 


E         F 


.  Typhoid  Bacillus 


E      H 


Colon  Bacillus 


Dysentery  Bacillus 
FIG.   5. 

sesses  considerable  protoplasm  A,  which  is  specific 
for  the  typhoid  bacillus,  that  it  possesses  also  certain 
protoplasm  B,  which  is  common  to  it,  and  to  the 

1   Park   and  Collins,  Journ.  Medical  Research,  Vol.  vii,  1904. 


AGGLUTININS  43 

colon  bacillus,  and  some  protoplasm  C,  common 
perhaps  to  some  other  bacterium.  In  the  case  of 
the  colon  bacillus,  protoplasm  D  is  specific,  i.e., 
possessed  only  by  this  bacillus,  while  B  is  common 
to  it  and  the  typhoid  bacillus,  and  E  common  to 
colon  and  dysentery  bacilli. 

By  immunization  with  the  typhoid  bacillus  we 
would  obtain  a  serum  containing  agglutinins  against 
protoplasm  A,  B,  and  C.  By  virtue  of  this  the 
serum  would  exert  some  agglutinating  power  also 
on  colon  bacilli.  On  extracting  such  a  serum  with 
the  typhoid  bacilli,  all  the  agglutinating  power  would 
be  lost,  that  for  the  typhoid  bacilli  as  well  as  that 
for  the  colon.  On  extracting  this  serum  with  the 
colon  bacilli  we  would  remove  the  agglutinating 
power  for  these  bacilli,  but  leave  the  specific  agglu- 
tinating power  on  typhoid  bacilli. 

Formation  of  the  Agglutinins  According  to  the 
Side-Chain  Theory  — •  Receptors  of  First,  Second  and 
Third  Order.  —  Ehrlich's  theory  as  outlined  in  the 
preceding  chapter  offers  a  ready  explanation  for  the 
development  of  these  bodies.  Certain  peculiarities 
of  the  agglutinins  require  merely  a  slight  elabora- 
tion of  detail  in  order  to  be  clearly  understood. 
According  to  Ehrlich  the  prime  function  of  the  side 
chains  of  a  cell  is  to  provide  for  the  nutrition  of  the 
cell.  Obviously  the  simplest  mechanism  for  this 
purpose  will  be  a  side  chain  which  merely  anchors 
the  food  molecule,  leaving  the  digestion  entirely  to 


44  IMMUNE  SERA 

the  cell  proper.  This  type  of  receptor  suffices  for 
comparatively  small  molecules  such  as  those  of  the 
toxins,  for  these  are,  after  all,  but  the  products  of 
cellular  activity.  When  the  protoplasm  of  the 
bacterial  cell  itself,  however,  is  to  serve  as  food  for 
the  animal  cell  the  latter  needs  more  than  a  mere 
anchoring  group,  it  needs  also  an  active  group 
which  can  in  some  way  act  on  the  huge  food  par- 
ticle and  make  it  more  readily  assimilable.  Such 
receptors  then  possess  two  groups,  a  haptophore 
group  and  another  functional  group  acting  on  the 
food  particle  thus  anchored.  Ehrlich  calls  these 
his  "  receptors  of  the  second  order,"  and  places  in 
this  class  the  agglutinins  and  the  precipitins.  The 
same  action  can  perhaps  be  more  economically 
brought  about  by  having  these  receptors,  in  addi- 
tion to  their  specific  haptophore  group,  possess  the 
means  by  which  the  action  of  a  ferment-like  sub- 
stance can  be  brought  to  bear  on  the  anchored 
food  particle.  Such  a  receptor  would  then  possess 
two  haptophore  groups,  one  for  the  food  particle, 
the  other  for  the  ferment-like  substance.  These 
are  Ehrlich's  "  receptors  of  the  third  order  "  and 
will  be  discussed  in  the  next  chapter.  Confining 
ourselves  for  the  present  to  the  agglutinins  we  find 
that  the  existence  of  the  two  groups  (haptophore 
and  agglutinating)  has  experimental  confirma- 
tion. We  have  seen  that  an  agglutinin  may  be 
changed  by  the  action,  for  instance,  of  acids,  so  that 


FIG.  6. — THE    VARIOUS    TYPES    OF     RECEPTORS    ACCORDING    TO 
EHRLICH. 

I.  Receptors  of  the  First  Order.  —  This    type  is   pictured    in  a.      The 

portion  e  represents  the  haptophore  group,  whilst  b  represents  a 
toxin  molecule,  which  possesses  a  haptophore  group  c  and  a 
toxophore  group  d.  This  represents  the  union  of  toxin  and 
antitoxin,  or  ferment  and  antiferment,  the  union  between  anti- 
body and  the  toxin  or  ferment  being  direct. 

II.  Receptors  of  the  Second  Order  are  pictured  in  c.      Here  e  represents 

the  haptophore  group,  and  d  the  zymophore  group  of  the 
receptor,  f  being  the  food  molecule  with  which  this  receptor 
combines.  Such  receptors  are  possessed  by  agglutinins  and 
precipitins.  It  is  to  be  noted  that  the  zymophore  group  is  an 
integral  part  of  the  receptor. 

III.  Receptors  of  the   Third  Order   are    pictured   in    III,  e  being  the 

haptophore  group  and  g  the  complementophile  group  of  the 
receptor.  The  complement  k  possesses  a  haptophore  group  h 
and  zymotoxic  group  2;  whilst  _/"  represents  the  food  molecule 
which  has  become  linked  to  the  receptor.  Such  receptors  are 
found  in  haemolysins,  bacteriolysins,  and  other  cytolysins,  the 
union  with  these  cellular  elements  being  effected  by  the  ambo- 
ceptor  (a  thrust-off  receptor  of  this  order).  It  is  to  be  noted 
that  the  digesting  body,  the  complement,  is  distinct  from  the 
receptor,  a  point  in  which  these  receptors  therefore  differ  from 
those  of  the  preceding  order. 


46  IMMUNE    SERA 

it  will  no  longer  possess  any  agglutinating  action, 
but  will  still  combine  with  the  bacteria.  In  other 
words,  the  agglutinating  group  has  been  lost,  the 
haptophore  has  remained  intact.  Once  the  agglu- 
tinating power  is  lost  it  cannot  be  restored,  in  which 
respect  the  agglutinins  differ  from  the  bacterio- 
lysins. 


BACTERIOLYSINS    AND    H^MOLYSINS 

Historical.  —  As  far  back  as  1874,  Gscheidlen 
and  Traube  l  demonstrated  that  considerable  quan- 
tities of  septic  material  could  be  injected  into  the 
circulation  of  warm-blooded  animals  without 
apparently  any  effect  on  the  animal.  Very  little 
was  thought  of  this  observation  at  the  time,  and  it 
is  not  until  more  than  ten  years  later  that  we  find 
a  similar  observation  made  by  Fodor.2  In  1888 
Nuttall 3  showed  that  normal  blood  serum  possessed 
marked  germicidal  properties,  and  his  observations 
stimulated  a  number  of  workers  who  undertook  to 
determine  the  conditions  most  favorable  to  the 
exhibition  of  this  phenomenon,  and  further  to 
decide  upon  the  constituent  of  the  serum  to  which 
this  property  was  due  or  whether  it  was  a  function 
of  the  serum  as  a  whole.  In  1899  Buchner  4  pub- 
lished a  series  of  experiments  and  showed  that  an 
exposure  of  55°C.  robs  the  serum  of  its  bacteri- 
cidal property.  He  also  concluded  that  the  active 
element  in  the  process  was  a  living  albumin  and 

1  Gscheidlen    and    Traube.    Schlez.     Gesellschaft.  f.    Vater- 
land.     Cultur,    Med.   Sect.,    1874. 

2  Fodor,  Deutsche  med.  Wochenschr,  1886. 

3  Nutall,  Zeitschr.  f.   Hygiene,  Vol.  iv,   1888. 

4  Buchner,  Centralblatt  Bacteriologie,  Vol.  v,  1889.     Archiv. 
f.  Hygiene,  Vol.  x,  1890. 

47 


48  IMMUNE  SERA 

suggested  for  it  the  name  "  alexin."  He  found  that 
it  was  possible  to  greatly  increase  the  bactericidal 
action,  (i.e.  the  quantity  of  "  alexin  ")  for  a  par- 
ticular bacterium  by  immunizing  an  animal  with 
that  bacterium. 

Pf eiffer's  Phenomenon.  —  An  enormous  advance 
in  the  study  of  immunity  was  made  in  the  dis- 
covery of  Pfeiffer's  phenomenon  in  1894,'  and  it  is 
to  Pfeiffer's  splendid  observations  *  that  we  owe 
the  first  and  most  important  insight  into  the  mode 
of  action  of  the  bacteriolytic  immune  sera.  A 
normal  guinea  pig  is  able  to  kill  and  dissolve  a 
number  of  living  cholera  bacilli  if  these  are  in- 
jected intraperitoneally.  If  in  such  an  animal  we 
gradually  increase  the  dose  injected,  it  will  be  pos- 
sible after  a  time  to  inject  at  one  dose  an  amount 
of  cholera  bacilli  that  represents  many  times  an 
ordinary  fatal  dose.  If  from  this  animal  we  now 
withdraw  serum  and  inject  it  into  another  animal, 
we  find  that  this  serum,  even  in  such  small  amounts 
as  the  fractional  part  of  a  centigram  or  even  of  a 
milligram,  is  able  to  protect  the  second  animal 
against  living  cholera  bacilli.  Under  the  influence 
of  these  small  amounts  of  serum  of  the  treated  ani- 
mal, the  organism  of  the  untreated  animal  is  able 
to  dissolve  large  amounts  of  cholera  bacilli,  amounts 
which  would  otherwise  be  invariably  fatal.  This 
process,  as  R.  Pfeiffer  showed,  is  a  specific  one,  i.e., 

1  R.  Pfeiffer,  Zeitschr.     Hygiene,  Vol.  xviii,  1894. 


BACTERIOLYS1NS    AND    HMMOLYSINS          49 

the  serum  of  the  guinea  pig  treated  with  cholera 
bacilli  transmits  an  increased  solvent  power  only 
for  cholera  bacilli,  but  not  for  any  other  species  of 
bacteria.  The  active  substance  of  such  a  bacterio- 
lytic  immune  serum  Pfeiffer  called  a  specific  bac- 
tericide.  If  we  allow  some  of  this  specific  cholera 
immune  serum  to  remain  for  some  time  outside  of 
the  body,  e.g.  in  a  bottle,  and  then  test  it  for 
solvent  properties  against  cholera  bacilli,  not  in  a 
living  body  but  in  a  test-tube,  we  shall  find  that  its 
power  is  almost  nil.  If  we  add  to  this  serum  in 
the  test-tube  some  fresh  peritoneal  exudate  or 
some  other  body  fluid,  such  as  serum  of  a  normal, 
untreated  guinea  pig,  as  Metchnikoff  first  did,  we 
find  that  this  serum  has  now  acquired  the  power 
to  rapidly  dissolve  cholera  bacilli  even  in  a  test-tube. 
Bordet,1  in  1895,  showed  that  in  order  for  the  specific 
immune  serum  to  dissolve  bacilli  in  a  test  tube, 
it  is  unnecessary  to  add  fresh  normal  serum  or 
peritoneal  fluid;  but  that  immune  serum  freshly 
drawn  from  the  vein  is  able  even  under  these  cir- 
cumstances to  dissolve  the  bacilli. 

Haemolysis.  —  Let  us  now  turn  for  a  moment  to 
the  development  of  this  subject  along  other  lines. 
If  we  go  back  to  the  time  when  blood  transfusion 
was  first  practised  we  find  it  stated  that  the  bloods 
of  different  animals  transfused  into  man  were  more 
or  less  directly  injurious,  and  not  capable  of  replac- 

1  Bordet,  Annal.  Inst.  Pasteur,   1895. 


50  IMMUNE  SERA 

ing  human  blood  for  this  purpose.  Landois  4  in  a 
study  published  in  1875  showed  that  while  trans- 
fusion of  a  foreign  blood  might  prove  fatal  to  an 
animal  the  transfusion  from  a  closely  related  species 
produced  no  ill  effects.  In  1898  Belfanti  and 
Carbone  2  showed  that  if  horses  were  injected  with 
red  blood  cells  of  rabbits,  the  serum  thereafter 
obtained  from  the  horses  would  have  acquired  an 
appreciable  toxicity  for  rabbits.  Shortly  after  this, 
Bordet  published  a  very  interesting  series  of  experi- 
ments. He  showed  that  the  serum  of  guinea  pigs 
after  these  had  been  injected  several  times  with  3 
to  5  cc.  of  defibrinated  rabbits'  blood  acquires  the 
property  to  dissolve  rapidly  and  intensely,  in  a 
test-tube,  the  red  blood  cells  of  a  rabbit;  whereas 
the  serum  of  a  normal  guinea  pig  is  incapable  of 
doing  this,  or  does  it  in  only  a  slight  degree.  Bordet 
could  further  show  that  this  action  is  a  specific  one, 
i.e.,  the  serum  of  animals  treated  with  rabbit  blood 
acquires  this  dissolving  property  only  for  the  red 
cells  of  rabbits,  not  for  those  of  any  other  species 
of  animal.  For  the  latter,  such  a  serum  is  no  more 
strongly  solvent  than  the  serum  of  a  normal  animal. 
The  same  property  that  Bordet  had  demonstrated 
in  the  serum  of  guinea  pigs  treated  with  rabbit 
blood  could  now  be  shown  for  the  sera  of  all  ani- 

1  Landois,  Zur  Lehre  von  der  Bluttransfusion,  Leipzig,  1875. 

2  Belfanti   and   Carbone,  Giorn.  della  R.  Acad.    di    Med.   di 
Torino,    1898. 


BACTERIOLYSINS    AND    HMMOLYS1NS          51 

mal  species  treated  with  blood  cells  of  a  different 
species.  We  can  formulate  this  as  follows:  The 
serum  of  animals,  species  A,  after  these  have  been 
injected  either  subcutaneously,  intraperitoneally, 
or  intravenously  with  erythrocytes  of  species  B, 
acquires  an  increased  solvent  action  for  erythro- 
cytes of  species  B,  and  only  for  this  species.1  It 
is  therefore  a  specific  action.  We  call  this  hcc- 
molysis,  and  the  substances  which  effect  the  solution 
of  the  red  cells,  hcemolysins  or  hcemotoxins. 

At  about  the  same  time,  and  independently  of 
Bordet,  similar  experiments  with  similar  results 
were  published  by  Landsteiner  2  and  v.  Dungern.3 
As  a  result  of  this  work,  the  acquired  toxicity  of 
horse  serum,  found  by  Belfanti  and  Carbone  when 
they  treated  horses  with  red  cells  of  rabbits,  was 
explained.  The  serum  of  the  horses  so  treated  had 
become  hcemolytic  for  rabbit  blood,  and  therefore 
caused  a  solution  or  destruction  of  the  red  cells 
in  the  living  body  just  as  it  did  in  a  test-tube. 

Nature  of  Hocmolytic  Sera.  —  In  a  subsequent 
study  Bordet 4  was  able  to  show  that  the  sol- 
vent power  of  the  specific  haemolysins  depended 
on  the  combined  action  of  two  constituents  of  the 
specific  serum.  When  the  fresh  hsemolytic  serum 
was  warmed  for  half  an  hour  to  55°C.,  it  lost  its 

1  We  shall  point  out  a  few  exceptions  later  on. 

2  Landsteiner,  Centralblatt  Bacteriol.      Vol.  xxv,  1899. 

3  Von   Dungern,    Munch,   med.    Wochenschrift,    1898, 
*  Bordet,  Annal.  Inst.  Pasteur,  Vol.  xii,  1898. 


52  IMMUNE   SERA 

power.  If  to  this  inactive  serum  a .  very  small 
amount  of  the  serum  of  a  normal  guinea  pig  was 
added  (a  serum  which  of  course  was  not  haemolytic 
for  rabbit  red  cells),  the  full  haemolytic  power  was 
restored  to  this  inactive  serum.  In  other  words, 
it  had  been  reactivated  by  this  addition. 

This  experiment  permits  of  only  one  conclusion, 
namely,  that  the  hasmolytic  action  of  the  specific 
haemolytic  serum  depends  on  two  substances.  One 
of  these  is  able  to  withstand  heating  to  55°C.,  and 
is  contained  only  in  the  specific  serum.  The  other 
is  destroyed  by  heating  to  55°C.,  and  is  contained 
not  only  in  the  specific  haemolytic  serum,  but  also 
in  the  serum  of  normal  untreated  animals. 

Buchner,  we  have  seen,  applied  the  term  alexins 
to  the  constituents  of  normal  serum  which  were 
actively  destructive  to  corpuscular  elements,  bac- 
teria, and  other  cells  with  which  they  came  in  con- 
tact. This  term  was  retained  by  Bordet  to  desig- 
nate that  constituent  of  normal  serum  which  did 
not  withstand  heating  to  55°  C.,  and  which  was  one 
of  the  factors  in  the  haemolytic  process.  The  other 
substance,  which  was  found  only  in  the  specific 
serum  and  which  withstood  heating  to  55°  C.,  he 
termed  substance  sensibilatrice. 

According  to  Bordet,  therefore,  the  substances 
required  for  haemolysis  are  the  substance  sensibila- 
trice of  the  specific  haemolytic  serurn  and  the 
alexin  which  exists  even  in  normal  serum.  The 


B ACT ERIOLY SINS    AND    H&MOLYSINS  53 

action  of  these  two  substances  Bordet  explains  by 
assuming  that  the  red  cell  is  not  vulnerable  to  the 
alexin ;  just  as,  for  example,  there  are  certain  sub- 
stances that  will  not  take  a  dye  without  the  previous 
action  of  a  mordant.  The  substance  sensibilatrice 
plays  the  role  of  mordant.  It  makes  the  blood 
cells  vulnerable  to  the  alexin,  so  that  the  latter  can 
attack  the  cells  and  dissolve  them.  The  alexin  he 
regards  as  a  sort  of  ferment  body  with  digestive 
powers. 

Bordet  says  further,  that  the  substance  sensi- 
bilatrice sensitizes  the  blood  cells  not  only  for  the 
alexin  derived  from  the  serum  of  the  same  species 
as  that  from  which  it  (the  substance  sensibilatrice) 
is  derived,  but  sensitizes  such  cells  also  for  the 
alexins  of  normal  sera  of  other  species.  For  ex- 
ample, in  the  foregoing  experiment  of  Bordet,  the 
substance  sensibilatrice  derived  from  the  guinea 
pig  by  treatment  with  rabbit  blood  sensitizes  the 
red  blood  cells  of  rabbits  not  only  for  the  alexin 
of  normal  guinea  pig  blood,  but  also  for  the  alexins 
of  other  normal  sera.  In  another  experiment 
this  author  showed  that  rabbit  red  cells  sensitized 
with  an  inactive  specific  haemolytic  serum  derived 
from  a  guinea  pig  would  dissolve  rapidly  on  the 
addition  of  normal  rabbit  blood.  Here,  then,  the 
rabbit  red  cells,  sensitized  (according  to  Bordet)  by 
the  substance  sensibilatrice  of  the  guinea  pig,  dissolve 
on  the  addition  of  the  alexin  of  their  own  serum. 


54  IMMUNE  SERA 

The  Exciting  Agent.  —  If  we  now  seek  to  discover 
the  constituent  part  of  the  red  cell  which  in  the 
treatment  excites  in  the  animal  body  the  production 
of  the  specific  haemolysin,  we  find  this  to  be,  accord- 
ing to  Bordet  and  v.  Dungern,  the  stroma  of  the  red 
cells.  This  separated  from  the  cell  contents  and 
injected  into  animals  will  likewise  excite  the  produc- 
tion of  specific  haemolytic  serum.  In  opposition 
to  this,  Nolf  assumes  that  the  stroma  excites  the 
production  of  the  above-mentioned  agglutinins,  and 
that  the  production  of  the  substance  sensibilatrice 
is  called  forth  by  the  contents  of  the  red  cells. 

Resume.  —  Reviewing  the  important  facts  we  have 
learned,  we  find  them  to  be  as  follows:  By  means 
of  the  treatment  of  one  species  of  animal  with  the 
red  cells  of  a  different  one,  the  serum  of  the  first 
species  acquires  an  uncommonly  increased  power 
to  dissolve  and  to  agglutinate  the  red  cells  of  the 
second  species.  This  increased  hasmolytic  power 
shows  itself  not  only  in  vivo,  so  that  an  animal 
so  treated  is  able  to  cause  red  cells  injected  into 
it  to  rapidly  dissolve  and  disappear,  but  it  shows 
itself  also  in  vitro  when  the  serum  of  this  animal 
is  used.  The  process  consists  in  the  combined 
action  of  two  substances,  that  which  is  excited 
in  response  to  the  injection,  the  substance  sensi- 
bilatrice, and  the  alexin  of  normal  serum. 

Analogy  between  the  Bacteriolytic  and  Haemoly- 
tic Processes.  —  If  we  now  recall  the  main  points  in 


BACTERIOLYSINS    AND    H^EMOLYSINS          55 

cholera  immunity  the  close  analogy  between  this  and 
the  subject  of  haemolysis  is  apparent.  Just  as,  when 
immunizing  an  organism  against  cholera  bacilli 
the  organism  responds  with  an  increased  solvent 
power  for  those  bacteria,  so  does  the  organism 
respond  when  it  is  treated,  i.e.  immunized,  with 
red  cells  of  another  species,  by  increasing  the  sol- 
vent power  of  its  serum  for  those  particular  cells. 
Furthermore,  just  as  the  hoemolytic  process  was 
seen  to  depend  on  the  combined  action  of  two  sub- 
stances, one  developed  in  the  hasmolytic  serum, 
the  other  already  present  in  normal  serum,  so  also 
in  the  bactericidal  process  just  studied  there  are 
two  factors.  It  is  easy  to  understand,  therefore, 
what  formerly  was  not  at  all  clear,  why  a  specific 
bactericidal  serum  against  cholera,  typhoid,  or 
other  infectious  disease  should  not  act  in  a  test- 
tube  unless  there  had  first  been  added  some  normal 
serum  (according  to  Metchnikoff),  or  there  had 
been  employed  a  perfectly  fresh  serum  (according 
to  Bordet):  simply  because  in  either  of  these 
ways  the  alexin  necessary  to  co-operate  with  the 
substance  sensibilatrice  is  introduced.  This  alexin 
no  longer  exists  in  the  immune  serum,  if  this  be 
not  perfectly  fresh,  for  we  have  seen  that  it  decom- 
poses either  on  warming,  or  spontaneously  on  stand- 
ing. A  bactericidal  serum,  therefore,  that  has 
stood  for  some  time  is  incapable  of  dissolving 
bacteria.  It  is  possible,  however,  to  make  an  old 


5 6  IMMUNE  SERA 

inactive  serum  again  capable  of  dissolving  bacteria 
in  vitro  by  adding  a  little  fresh  alexin,  according 
to  the  suggestion  of  Metchnikoff.  In  other  words, 
it  is  thus  reactivated.  Another  obscure  point  was 
cleared  up  by  these  studies:  why  a  specific  bac- 
tericidal serum  which  is  inactive  in  vitro  should 
be  intensely  active  in  the  living  body.  This  is 
because  in  the  living  body  the  serum  finds  the  alexin 
necessary  for  its  working,  which  is  not  the  case  in 
the  test-tube  unless  fresh  normal  serum  be  added. 
We  see  from  all  this  that  even  the  first  experiments 
in  haemolysis  have  served  to  clear  up  a  number  of 
practical  points  in  an  important  branch  of  bacteri- 
ology. 

Ehrlich  and  Morgenroth  on  the  Nature  of  Haemo- 
lysis. —  In  continuing  the  study  of  haemolysins  we 
must  note  particularly  the  researches  of  Ehrlich 
and  Morgenroth.1  These  authors  asked  themselves 
the  following  questions:  (i)  What  relation  does 
the  haemolytic  serum  or  its  two  active  components 
bear  to  the  cell  to  be  dissolved?  (2)  On  what 
does  the  specificity  of  this  haemolytic  process 
depend  ?  Ehrlich  was  led  to  these  researches  partic- 
ularly by  his  so-called  Side-chain  Theory,  which 
we  shall  examine  in  a  moment. 

He  made  his  experiments  with  a  haemolytic 
serum  that  had  been  derived  from  a  goat  treated 

1  Ehrlich  and  Morgenroth.  See  the  various  papers  in  "Col- 
lected Studies  on  Immunity,"  Wiley  and  Sons,  New  York,  1906. 


BACTERIOLYSINS  AND  H&MOLYSINS  $? 

with  the  red  cells  of  a  sheep.  This  serum,  there- 
fore, was  hsemolytic  specifically  for  sheep  blood 
cells;  i.e.,  it  had  increased  solvent  properties  exclu- 
sively for  sheep  blood  cells. 

Basing  his  reasoning  on  his  side-chain  theory, 
Ehrlich  argued  as  follows:  "  If  the  haemolysin  is 
able  to  exert  a  specific  solvent  action  on  sheep 
blood  cells,  then  either  of  its  two  factors,  the  sub- 
stance sensibilatrice  of  Bordet  or  the  alexin  of  nor- 
mal serum,  must  possess  a  specific  affinity  for  these 
red  cells.  It  must  be  possible  to  show  this  experi- 
mentally." Such  in  fact  is  the  case,  and  the  experi- 
ments devised  by  him  are  as  follows : 

Experiment  i. —  Ehrlich  and  Morgenroth,  as 
already  said,  experimented  with  a  serum  that  was 
specifically  hasmolytic  for  sheep  blood  cells.  They 
made  this  inactive  by  heating  to  55°  C.,  so  that  then 
it  contained  only  the  substance  sensibilatrice. 
Next  they  added  a  sufficient  quantity  of  sheep 
red  cells,  and  after  a  time  centrifuged  the  mixture. 
They  were  now  able  to  show  that  the  red  cells  had 
combined  with  all  the  substance  sensibilatrice,  and 
that  the  supernatant  clear  liquid  was  free  from  the 
same.  In  order  to  prove  that  such  was  the  case 
they  proceeded  thus :  To  some  of  the  clear  centri- 
fuged fluid  they  added  more  sheep  red  cells;  and, 
in  order  to  reactivate  the  serum,  a  sufficient  amount 
of  alexin  in  the  form  of  normal  serum  was  also 
added,  The  red  cells,  however,  did  not  dissolve  — 


$8  IMMUNE   SERA 

there  was  no  substance  sensibilatrice.  The  next 
point  to  prove  was  that  this  substance  had  actually 
combined  with  the  red  cells.  The  red  cells  which 
had  been  separated  by  the  centrifuge  were  mixed 
with  a  little  normal  salt  solution  after  freeing  them 
as  much  as  possible  from  fluid.  Then  a  little  alexin 
in  the  form  of  normal  serum  was  added.  After 
remaining  thus  for  two  hours  at  37°  C.  these  cells 
had  all  dissolved. 

In  this  experiment,  therefore,  the  red  cells  had 
combined  with  all  the  substance  sensibilatrice, 
entirely  freeing  tlje  serum  of  the  same.  That  the 
action  was  a  chemical  one  and  not  a  mere  absorp- 
tion was  shown  by  the  fact  that  red  blood  cells  of 
other  animals,  rabbits  or  goats  for  example,  exerted 
no  combining  power  at  all  when  used  instead  of 
the  sheep  cells  in  the  above  experiment.  The 
union  of  these  cells,  moreover,  is  such  a  firm  one 
that  repeated  washing  of  the  cells  with  normal  salt 
solution  does  not  break  it  up. 

The  second  important  question  solved  by  these 
authors  was  this:  What  relation  does  the  alexin 
bear  to  the  red  cells  ?  They  studied  this  by  means 
of  a  series  of  experiments  similar  to  the  preceding. 

Experiment  2.  —  Sheep  blood  was  mixed  with 
normal,  i.e.  not  haemolytic,  goat  serum.  After  a 
time  the  mixture  was  centrifuged  and  the  two  por- 
tions tested  with  substance  sensibilatrice  to  deter- 
mine the  presence  of  alexin.  It  was  found  that  in 


OF 

IFO_ 
BACTER1OLYS1NS  AND  H^EMOLYSINS 

this  case  the  red  cells  acted  quite  differently.  In 
direct  contrast  to  their  behavior  toward  the  sub- 
stance sensibilatrice  in  the  first  experiment,  they 
now  did  not  combine  with  even  the  smallest  por- 
tion of  alexin,  and  remained  absolutely  unchanged. 
Experiment  3. --The  third  series  of  experiment 
was  undertaken  to  show  what  relations  existed 
between  the  blood  cells  on  the  one  hand,  and  the 
substance  sensibilatrice  and  the  alexin  on  the 
other,  when  both  were  present  at  the  same  time, 
and  not,  as  in  the  other  experiments,  when  they 
were  present  separately.  This  investigation  was 
complicated  by  the  fact  that  the  specific  immune 
serum  very  rapidly  dissolves  the  red  cells  for  which 
it  is  specific,  and  that  any  prolonged  contact  be- 
tween the  cells  and  the  serum,  in  order  to  effect 
binding  of  the  substance  sensibilatrice,  is  out  of 
the  question.  Ehrlich  and  Morgenroth  found  that 
at  o°  C.  no  solution  of  the  red  cells  by  the  haemo- 
lytic  serum  takes  place.  They  therefore  mixed  some 
of  their  specific  haemolytic  serum  with  sheep  blood 
cells,  and  kept  this  mixture  at  o°-3°  C.  for  sev- 
eral hours.  No  solution  took  place.  They  now 
centrifuged  and  tested  both  the  sedimented  red 
cells  and  the  clear  supernatant  serum.  It  was 
found  that  at  the  temperature  o°-3°  C.  the  red 
cells  had  combined  with  all  of  the  substance  sen- 
sibilatrice, but  had  left  the  alexin  practically 
untouched. 


60  IMMUNE  SERA 

It  still  remained  to  show  the  relation  of  these 
two  substances  to  the  red  cells  at  higher  temper- 
atures. At  37°-4o°  C.,  as  already  mentioned, 
haemolysis  occurs  rapidly,  beginning  usually  within 
fifteen  minutes.  It  was  possible,  therefore,  to 
leave  the  cells  and  serum  in  contact  for  not  over 
ten  minutes.  Then  the  mixture  was  centrifuged 
as  before.  The  sedimented  blood  cells  mixed  with 
normal  salt  solution  showed  haemolysis  of  a  moder- 
ate degree.  The  solution  became  complete  when 
a  little  normal  serum  was  added.  The  supernatant 
clear  fluid  separated  by  the  centrifuge  did  not  dis- 
solve sheep  red  cells.  On  the  addition,  however, 
of  substance  sensibilatrice  it  dissolved  them  com- 
pletely. 

So  far  as  concerns  the  technique  of  the  experi- 
ments, I  should  like  to  observe  that  the  addition 
of  red  cells  in  this  as  well  as  in  all  the  following 
experiments  was  always  in  the  form  of  a  5%  mix- 
ture or  suspension  in  0.85%,  i.e.  isotonic,  salt  solu- 
tion. 

The  significance  of  the  last  of  the  above-cited 
experiments  is  at  once  apparent.  It  is  that  the 
substance  sensibilatrice  possesses  one  combining 
group  with  an  intense  affinity  (active  even  at  o°  C.), 
for  the  red  cell,  and  a  second  group  possessing  a 
weaker  affinity  (one  requiring  a  higher  temperature) 
for  the  alexin. 

Nomenclature.  -  -  In  place  of  the  name  substance 


BACTERIOLYSINS  AMD  H^MOLYSINS          6l 

sensibilatrice  Ehrlich  first  introduced  the  term 
immune  body,  later  on  he  called  it  the  amboceptor. 
In  the  following  pages  we  shall  use  the  term  immune 
body,  as  this  had  already  been  used  by  R.  Pfeiffer 
to  designate  the  same  substance  in  bactericidal 
serum.  Other  names  proposed  for  this  substance 
have  been  substance  fixatrice  by  Metchnikoff,  copula, 
desmon,  preparator  by  Miiller.  Instead  of  the  name 
alexin,  Ehrlich  now  uses  the  term  complement  in  order 
to  express  the  idea  that  this  body  completes  the 
action  of  the  immune  body. 

In  contrast  to  the  specific  affinity  which  the  red 
cells  possess  for  the  immune  body,  these  cells  pos- 
sess no  affinity  whatever  for  the  alexin,  as  has  been 
shown  by  the  second  of  Ehrlich 's  experiments. 
The  alexin,  therefore,  possesses  no  combining  group 
which  can  attach  itself  directly  to  the  red  blood 
cell.  It  acts  on  these  cells  only  through  an  inter- 
mediary, the  immune  body,  which  therefore  must 
possess  two  binding  groups  one  which  attaches  to 
the  red  blood  cell  and  the  other  to  the  alexin  of 
normal  serum.  As  already  stated,  the  group 
which  attaches  to  the  red  blood  cell  possesses  a 
much  stronger  affinity  than  that  which  combines 
with  the  alexin „  This  follows  from  the  last  two 
experiments  of  Ehrlich  before  cited,  in  which  he 
showed  that  at  the  lower  temperature,  and  with 
both  substances  present  with  the  blood  cells,  only 
the  immune  body  combined  with  the  cells,  while 


62  IMMUNE  SERA 

the  alexin  remained  uncombined.  At  the  higher 
temperature  the  alexin  also  exerted  its  affinity,  for 
then  the  red  cells  combined  with  all  the  immune 
body  and  with  part  of  the  alexin.  We  saw  that 
after  a  time  the  red  cells  partially  dissolved,  but 
that  complete  solution  occurred  only  after  some 
fresh  alexin  had  been  added.  This  showed  that 
although  the  red  cells  had  combined  with  all  the 
immune  body  necessary  for  their  solution,  they  had 
been  unable  to  bind  all  the  alexin  necessary.  We 
may  say,  therefore,  that  that  group  of  the  immune 
body  which  combines  with  the  red  cell  has  a 
stronger  affinity  than  that  which  combines  with  the 
alexin. 

Role  of  the  Immune  Body.  —  According  to  Ehrlich, 
then,  the  role  of  the  immune  body  consists  in  this, 
that  it  attaches  itself  to  the  red  cell  on  the  one  hand, 
and  to  the  complement  on  the  other,  and  in  this  way 
brings  the  digestive  powers  of  the  latter  to  bear 
upon  the  cell,  the  complement  possessing  no  affinity 
for  the  red  cell.  Immune  body  and  complement 
have  no  very  great  affinity  for  each  other.  At  o°  C. 
they  may  exist  in  serum  side  by  side,  and  they 
combine  only  at  higher  temperatures. 

The  amount  of  immune  body  which  combines 
with  the  red  cells  may  vary  greatly,  as  the  experi- 
ments of  Bordet  and  of  Ehrlich  clearly  show. 
Some  red  cells  combine  with  only  just  enough 
immune  body  to  effect  their  solution.  Others  are 


BACTERIOLYSINS  AND  H&MOLYSINS          63 

able  to  so  saturate  themselves  with  immune  body 
that  they  may  have  a  hundred  times  the  amount 
necessary  for  their  solution. 

On  what  the  Specificity  Depends.  —  From  the  pre- 
ceding it  follows  that  the  specific  action  of  the 
haemolytic  sera,  and,  I  may  at  once  add,  of  the  bac- 
tericidal sera  also,  is  due  exclusively  to  the  immune 
body.  This  possesses  a  combining  group  which  is 
specific  for  the  cells  with  which  the  animal  was 
treated;  e.g.,  the  combining  group  of  an  immune 
body  produced  by  treatment  with  rabbit  blood 
will  fit  only  to  a  certain  group  in  the  blood  cells  of 
rabbits;  an  immune  body  produced  by  treatment 
with  chicken  blood  will  fit  only  to  parts  of  the  red 
cells  of  chickens;  one  produced  by  treating  an  ani- 
mal with  cholera  bacilli  will  fit  only  to  this  species 
of  bacteria  and  combine  only  with  the  members  of 
it.  Keeping  to  the  well-known  simile  of  Emil 
Fischer,  the  relation  is  like  that  between  lock  and 
key,  each  lock  being  fitted  only  by  a  particular 
key. 

To  repeat  —  for  the  point  is  of  the  greatest 
importance  —  the  role  of  the  immune  body  consists 
in  tying  the  complements  of  normal  serum,  which 
have  no  affinity  for  the  red  cells  or  for  the  bacteria, 
indirectly  to  these  cells  so  that  their  solution  and 
digestion  may  be  effected  by  the  complements. 
In  other  words,  the  immune  body  serves  to  con- 
centrate on  the  corpuscular  element  to  be  dis- 


64 


IMMUNE  SERA 


solved  all  the  widely  distributed  complement  found 
in  normal  serum. 

The  relation  existing  between  complement,  im- 
mune body  (i.e.,  amboceptor)  and  erythrocyte  is 
shown  in  the  accompanying  figure  reproduced 
after  Levaditi,  a  pupil  of  Ehrlich. 


n. 


COMPLEMENT- 


IMMUNE  BODY- 


CELL— a 


— zymotoxic  group 

— hatophore  group 

— complementophile  gr. 

— cytophile  group 
— receptor 


FIG.  7 

Difference  between  a  Specific  Serum  and  a  Normal 
One.  -  -  The  difference,  then,  between  a  specific 
haemolytic  or  a  specific  bactericidal  serum  and  a 
normal  one  consists  in  this  —  that  the  specific  serum 
contains  an  immune  body  which  is  specific  for  a 
certain  cellular  element  and  by  means  of  which  the 
complement  present  in  all  normal  serum  can  be  con- 
centrated on  this  element  to  cause  its  solution.  We 
shall  return  to  this  subject  later. 

Diverging  Views  of  Ehrlich  and  Bordet.  —  Now  if 
we  recall  the  first  experiments  of  Bordet  and  his 
conclusions  respecting  the  manner  in  which  the 
factors  concerned  acted,  we  shall  at  once  see  how 


BACTERIOLYSINS  AND  H^MOLYSINS          65 

Ehrlich  and  Bordet  differ,  Bordet  assumes  that 
the  substance  sensibilatrice  (the  immune  body) 
acts  as  a  kind  of  mordant  on  the  red  cells  or  bac- 
teria, sensitizing  these  to  the  action  of  the  alexin 
(complement).  According  to  Ehrlich,  however,  the 
process  is  not  analogous  to  a  staining  process, 
but  follows  definite  laws  of  chemical  combination, 
there  being,  in  fact,  no  affinity  whatever  between 
the  complement  and  the  blood  cells  or  bacteria. 
Furthermore,  according  to  this  authority,  the  com- 
plement always  acts  through  the  mediation  of  the 
immune  body,  which  possesses  two  combining 
groups;  one,  the  cytophile  group,  combining  with 
the  cell,  and  another,  the  complementophile  group, 
combining  with  the  complement.  Both  observers 
have  devised  a  series  of  ingenious  experiments  to 
support  their  views.  But  as  these  can  interest  only 
the  specialist,  we  shall  omit  their  discussion  here. 
For  such  details  the  original  articles  may  be  con- 
sulted. 

The  Side-Chain  Theory  Applied  to  these  Bodies.  - 
All  of  the  specific  relations  which,  in  a  previous 
chapter,  we  saw  existed  between  toxin  and  anti- 
toxin, Ehrlich  and  Morgenroth  in  their  experi- 
ments above  noted  found  existed  also  between 
immune  body  and  the  specific  blood  cell.  The 
immune  body  must  therefore  possess  a  haptophore 
group  which  fits  exactly  to  certain  receptors  or 
side  chains  of  the  red  cells,  just  as  the  anti-body 


66  IMMUNE  SERA 

according  to  the  side-chain  theory  possesses  a 
group  that  fits  exactly  into  the  specific  combining 
group  —  i.e.,  haptophore  group  —  of  the  toxin  or 
toxoid  used  for  exciting  the  immunity. 

If,  for  example,  we  produce  a  haemolytic  serum 
specific  for  red  cells  of  a  rabbit  by  injecting  an 
animal  with  these  cells,  the  haptophore  groups  of 
this  serum,  i.e.,  the  free  side  chains  thrust  off,  must 
possess  specific  combining  relations  with  the  red 
cells  of  rabbits.  That  such  is  the  case  in  the  haemo- 
lytic immune  serum  we  saw  from  the  experiments 
of  Ehrlich  and  Morgenroth. 

In  consequence  of  all  this,  Ehrlich  widened 
the  application  of  his  side-chain  theory  so  as  to 
include  not  only  the  production  of  antitoxin  but 
also  the  production  of  bactericidal,  haemolytic, 
and  other  immune  bodies.  He  expressed  this 
somewhat  as  follows:  //  any  substance,  be  it  toxin, 
ferment,  constituent  of  a  bacterial  or  animal  cell,  or 
of  animal  fluid,  possess  the  power  by  means  of  a 
fitting  haptophore  group  to  combine  with  side  chains 
(receptors)  of  the  living  organism,  the  possibility  for 
the  overproduction  and  throwing  off  of  these  recep- 
tors is  given,  i.e.,  the  possibility  to  produce  a  cor- 
responding anti-body. 

Specific  anti-bodies  in  the  serum  as  a  result  of 
immunizing  processes  can  only  be  produced,  there- 
fore, by  substances  which  possess  a  haptophore 
group  and  which,  in  consequence,  are  able  to  form  a 


BACTERIOLYSINS  AND  HMMOLYSINS          6? 

firm  union  with  a  definite  part  of  the  living  or- 
ganism, the  receptor.  This  is  not  the  case  with 
alkaloids,  e.g.,  morphine,  strychnine,  etc.,  which 
according  to  Ehrlich  enter  into  a  loose  union,  a  kind 
of  solid  solution  with  the  cells.  It  is  for  this  reason 
that  we  are  unable  to  produce  any  anti-bodies  in 
the  blood  serum  against  these  poisons.  Ehrlich 
says  further  that  all  of  the  substances  taking  part 
in  the  production  of  immunity,  including  of  course 
complement  and  immune  body,  have  certain  defi- 
nite affinities  for  each  other,  and  in  order  to  act 
they  must  fit  stereochemically  to  each  other. 

As  we  have  already  seen,  we  are  able  by  means 
of  the  injection  of  a  variety  of  substances  or  cells 
to  produce  a  similar  variety  of  immune  bodies  in 
the  serum.  Thus  we  can  immunize  a  rabbit  so 
that  its  serum  will  possess  specific  haemolytic 
bodies  against  the  red  cells  of  guinea  pigs,  goats, 
chickens,  and  oxen  and  specific  bactericidal  bodies 
against  cholera  and  typhoid  bacilli,  etc.,  and  as  we 
shall  see,  still  other  groups  of  an ti -bodies. 

Multiplicity  of  Complements.  —  Under  these  cir- 
cumstances an  important  question  presents  itself: 
Is  there  in  normal  serum  one  single  complement 
which  completes  the  action  of  all  these  various 
immune  bodies,  one,  for  example,  which  in  the 
above  illustration  will  fit  all  the  haemolytic  immune 
bodies  as  well  as  all  the  bactericidal  ones,  or 
are  there  a  great  many  different  complements? 


68  IMMUNE  SERA 

Ehrlich,  as  a  result  of  his  experimental  work 
with  Morgenroth,  claims  that  the  latter  is  the  case ; 
namely,  that  it  takes  a  different  complement  to  fit 
the  immune  body  specifically  haemolytic  for  guinea 
pig  blood  than  it  does  to  fit  that  specific  for  chicken 
blood. 

Bordet,  on  the  other  hand,  assuming  that  the 
immune  body  plays  the  role  of  mordant,  believes 
as  does  also  Buchner,  that  there  is  but  one  single 
complement  in  the  serum.  According  to  him, 
this  complement  is  able  to  dissolve  blood  cells  as 
well  as  bacteria  after  these  have  been  sensitized 
by  their  specific  immune  body.  Each  of  these 
authors  supports  his  claims  by  means  of  ingenious 
experiments,  for  the  details  of  which,  however, 
we  must  refer  to  the  original  articles,  as  they  require 
the  knowledge  of  a  specialist  for  their  compre- 
hension. We  shall,  however,  give  one  of  Bordet 's  l 
experiments  on  this  point  in  some  detail  since  it  has 
found  extensive  application  in  another  direction: 

The  Bordet-Gengou  Phenomenon.  —  Bordet  sensi- 
tized blood  corpuscles  with  appropriate  amboceptors, 
and  then  exposed  them  to  the  action  of  a  freshly  drawn 
normal  serum.  If  now  he  waited  for  the  occurrence 
of  haemolysis  and  then  added  sensitized  cells  (bacteria 
or  blood  corpuscles  of  a  different  species),  the  latter  re- 
mained entirely  unchanged,  although  the  serum  that  had 
been  used  as  complement  was  capable  in  its  original  con- 

1  Bordet  and  Gengou,  Annal.  Inst.  Pasteur.     Vol.  xv,  1901. 


BACTERIOLYSINS  AND   HMMOLYSINS  69 

dition  of  destroying  these  also.  When  fresh  serum  was 
first  brought  into  contact  with  sensitized  bacteria,  simi- 
lar results  were  obtained.  The  blood  corpuscles  sub- 
sequently added  did  not  then  undergo  haemolysis. 
//  such  an  action  on  one  of  the  sensitive  substrata  has 
once  taken  place,  the  active  sera,  as  a  rule,  are  deprived 
of  all  their  complement  functions,  from  which  Bordet 
concludes  that  the  destruction  of  the  most  varied 
elements  by  one  and  the  same  serum  must  be  due  to  a 
single  complement. 

It  may  be  said  in  passing  that  Ehrlich  admits  the 
correctness  of  the  above  experimental  results,  but 
brings  forward  additional  arguments  showing  that 
Bordet's  interpretation  as  to  the  existence  of  only  a 
single  complement  cannot  be  accepted. 

This  experiment  of  Bordet  is  usually  spoken  of  as 
the  "  Bordet-Gengou  phenomenon  "  and  is  now  used 
largely  in  determining  whether  or  not  a  given  serum 
possesses  certain  amboceptors.  The  serum  t;>  be 
tested  is  first  heated  and  then  mixed  with  a  small 
quantity  of  fresh  normal  serum  (complement)  and 
with  an  emulsion  of  the  bacterium  whose  amboceptors 
it  is  desired  to  discover.  After  standing  for  six  hours 
at  room  temperature,  red  blood  cells  previously  treated 
with  heated  haemolytic  serum  are  added.  If  there  is 
no  haemolysis  it  is  held  to  mean  that  the  complement 
in  the  fresh  serum  which  was  suitable  for  lysis  of 
properly  prepared  blood  corpuscles,  has  been  absorbed 
by  the  bacteria  by  reason  of  the  presence  of  specific 
amboceptors  in  the  serum  tested. 

Wassermann 1  has  recently  successfully  applied  this 
method  in  measuring  the  amboceptor  content  of  specific 

1  Wassermann,  Neisser  and  Bruck,  Deutsche  med.  Wochen- 
schr,  1906;  Wassermann  and  Plaut,  Ibid. 


70  IMMUNE  SERA 

meningococcus  sera  and    also  in    diagnosing    syphilitic 
antigens  and  antibodies. 

Neisser  and  Sachs 1  have  recently  described  a  pro- 
cedure for  the  forensic  diagnosis  of  blood  stains.  The 
principle  of  this  is  the  same  as  in  the  preceding  although 
in  so  far  as  a  specific  precipitin  serum  is  made  use  of, 
the  procedure  is  really  modelled  after  the  "  Gengou- 
Moreschi  "  phenomenon. 

If  human  blood  serum  is  mixed  with  a  specific 
human  precipitin  serum  derived  from  rabbits,  it  will 
be  found  that  the  mixture  binds  complement.  Has- 
molysin  subsequently  added  is  unable  to  dissolve  its 
specific  red  blood  cells,  owing  to  this  locking  up  of 
the  complement.  Only  the  serum  of  monkeys  has  a 
similar  effect.  The  amount  required  is  extremely 
minute,  yiroVoo  "to  TooVo¥  cc-  human  blood  or  monkey 
blood  sufficing.  Extracts  of  human  blood  stains  will  also 
produce  the  desired  effect.  The  authors  believe  that 
the  immunization  with  human  blood  serum  gives  rise 
not  only  to  precipitins  but  also  to  amboceptors  which 
then  are  able  to  unite  with  their  corresponding  unformed 
albuminous  bodies  and  so  bind  complement.  Others 
are  of  the  opinion  that  the  complement  is  bound  by 
the  precipitin-precipitum  combination. 

The  test  is  extremely  delicate  and  has  been  found 
trustworthy  by  a  number  of  investigators.  In  view 
of  the  importance  of  such  tests  in  medico-legal  cases, 
Neisser  and  Sachs  suggest  that  it  should  always  be 
used  in  addition  to  the  well  known  Wassermann- 
Uhlenhuth  precipitin  test. 

Normal  Serum,  its  Haemolytic  and  Bacteriolytic 
Action.  —  Inquiring  now  into  the  essential  differ- 

1  Neisser  and   Sachs,    Berliner  klin   Wochenschrift,    1905. 


BACTER1OLYSINS  AND   H^EMOLYSINS 


ence  between  a  specific  haemolytic  or  bactericidal 
serum  and  a  normal  one,  we  must  first  of  all  study 
the  behavior  of  normal  serum  toward  foreign  red 
cells  and  bacteria.  It  has  long  been  known  to 
physiologists  that  fresh  normal  serum  of  many 
animals  has  the  power  to  dissolve  blood  cells  of 
another  species.  This  was  studied  especially  by 
Landois.  One-half  to  one  c.c.  of  normal  goat  serum, 
for  example,  is  able  to  dissolve  5  c.c.  of  a  5%  mix- 
ture (in  normal  salt  solution)  of  rabbit  or  guinea 
pig  red  cells.  In  the  same  way,  these  red  cells 
are  dissolved  by  the  sera  of  oxen,  of  dogs,  etc. 
This  normal  globidicidal  property  of  the  serum  cor- 
responds to  another  which  fresh  normal  serum  was 
found  to  possess,  namely,  the  property  to  dissolve 
appreciable  quantities  of  many  species  of  bacteria. 
This  analogy  was  pointed  out  by  Fodor,  Nutall, 
Nissen,  and  especially  by  Buchner.  We  call  this 
the  bactericidal  property  of  fresh  normal  serum. 

This   property    is   well    illustrated    by    the    following 
protocol  from  Park. 


No.  of  bacteria 
in  i  cc.  fluid. 

Amount  of 
serum  added. 

Approximate  number  alive  after  being  kept  at  37°  C* 

One  hour. 

Two  hours.    . 

Twenty-seven  hrs. 

30,000 
100,000 
1,000,000 

O.I   CC. 
O.I   CC. 
0.  I    CC. 

400 

5,000 
400,000 

2 
1,000 
2,000,000 

0 
2OO,OOO 
IO,OOO,OOO 

It  is  at  once  apparent  that  the  number  of  bacteria 
introduced  is  an  important  factor,  the  normal  serum 
being  able  to  kill  off  only  a  certain  number. 


72  IMMUNE   SERA 

Buchner,  as  we  have  already  seen,  had  studied 
this  bactericidal  action  carefully  and  ascribed  the 
action  to  a  substance  found  in  all  normal  serum, 
which  he  called  alexin.  According  to  his  experi- 
ments, this  is  a  very  unstable  substance,  decom- 
posing spontaneously  on  standing  or  on  heating  for 
a  few  minutes  to  55°  C.,  or  readily  on  the  action 
of  chemicals.  According  to  this  author  all  the 
globulicidal  and  bactericidal  functions  of  normal 
serum  are  performed  by  this  one  substance,  the 
alexin. 

Active  and  Inactive  Normal  Serum.  —  Ehrlich  and 
Morgenroth  now  took  up  the  study  of  the  haemo- 
lytic  action  of  normal  serum.  They  sought  par- 
ticularly to  discover  whether  in  normal  serum 
the  haemolytic  property  depended  on  the  action  of 
a  single  substance,  the  complement  (Buchner's 
alexin),  or  whether  here  as  in  the  specific  haemo- 
lytic  serum  it  depended  on  the  combined  action 
of  two  substances.  For  this  purpose  they  used 
guinea-pig  blood,  which  is  dissolved  by  normal 
dog  serum.  If  this  serum  was  heated  to  55°  C.,  it 
lost  its  haemolytic  power.  It  was  necessary  now 
to  show  that  in  this  inactive  dog  serum  there 
remained  a  second  substance  which  could  be  reacti- 
vated after  the  manner  of  reactivating  an  old 
specific  haemolytic  serum.  This  had  its  difficulties, 
for  they  could  not  add  normal  dog  serum.  This, 
as  we  saw,  is  already  haemolytic  for  guinea-pig 


BACTERIOLYSINS  AND  HMMOLYSINS  73 

blood.  "  Possibly,"  said  they,  "  there  exists  a  com- 
plement of  another  animal  which  will  fit  the  hypo- 
thetical second  substance  of  this  dog  serum." 
This  proved  to  be  the  case,  the  complement  of 
guinea-pig  blood  fulfilling  the  requirements.  If 
they  added  to  the  inactive  normal  dog  serum  about 
2  c.c.  normal  guinea-pig  serum  the  haemolytic  prop- 
erty was  restored  and  the  guinea-pig  red  cells 
dissolved  completely.  This  can  only  be  explained 
by  assuming  that  in  guinea-pig  blood  there  exists 
a  complement  which  happens  to  fit  the  hapto- 
phore  group  of  the  second  substance  or  inter-body, 
of  the  normal  dog  serum.  This  combination  of 
guinea-pig  blood,  inactive  normal  dog  serum,  and 
a  reactivating  normal  guinea-pig  serum  is  well 
adapted  to  demonstrate  the  existence  in  normal 
dog  serum  of  an  inter-body;  for  the  guinea-pig 
serum  should  be  the  best  possible  preservative  for 
the  guinea-pig  red  cells.  The  haemolysis  following 
the  addition  of  this  serum  shows  positively  the  exist- 
ence of  a  substance  in  the  dog  serum  which  has 
acted  with  something  in  the  guinea-pig  serum.1 

1  Of  such  combinations,  i.e.,  combinations  in  which  a  com- 
plement derived  from  the  same  animal  from  which  the  red  cells 
are  derived  fits  to  the  inter-body  of  other  species  of  animals, 
causing  the  solution  of  red  cells  of  the  latter,  Ehrlich  and 
Morgenroth  found  still  other  examples.  For  instance,  guinea- 
pig  blood,  inactive  calf  serum,  guinea-pig  serum;  goat  blood, 
inactive  rabbit  blood,  goat  serum;  sheep  blood,  inactive  rabbit 
blood,  sheep  serum;  guinea-pig  blood,  inactive  sheep  serum, 
guinea-pig  serum. 


74  IMMUNE   SERA 

Inter-body  and  Complement.  --We  see,  then,  that 
the  haemolytic  action  of  normal  sera  depends,  just 
as  that  of  the  specific  hasmolytic  sera,  on  the  com- 
bined action  of  two  bodies:  one,  the  inter-body, 
which  corresponds  to  the  immune  body  of  the 
specific  sera,  and  a  second  or  complement.  In 
speaking  of  the  constituents  of  normal  serum, 
Ehrlich  and  Morgenroth  prefer  to  use  this  term 
inter-body  to  distinguish  it  from  the  immune  bodies 
of  specific  haemolytic  sera. 

Action  not  Entirely  Specific.  —  It  has  also  been 
found  that  there  frequently  exist  normal  sera  which 
are  haemolytic  not  only  for  one  species  of  red  cell, 
but  for  several.  We  saw,  for  instance,  that  normal 
goat  serum  dissolved  the  red  cells  of  guinea  pigs 
and  rabbits.  The  question  now  arises,  Is  this  prop- 
erty of  normal  goat  serum  due  to  two  inter-bodies 
existing  in  the  serum  side  by  side,  one  fitting  the 
red  cells  of  the  guinea  pig,  the  other  those  of  the 
rabbit?  Ehrlich  and  Morgenroth  answered  this 
in  the  affirmative,  for  in  the  following  experi- 
ment they  succeeded  in  having  each  of  the  two 
inter-bodies  combine  with  its  respective  cell.  To 
some  inactive  normal  goat  serum  they  added  rab- 
bit blood  and  centrifuged  the  mixture.  To  the 
separated  clear  fluid  they  again  added  some  rab- 
bit red  cells  as  well  as  normal  horse  serum  to  reac- 
tivate the  mixture.  Horse  serum  is  not  haemo- 
lytic for  rabbit  red  cells.  The  mixture  remained 


BACTERIOLYSINS  AND  H&MOLYSINS  75 

unchanged,  no  haemolysis  taking  place.  If,  how- 
ever, they  added  some  of  this  normal  horse  serum 
to  the  centrifuged  red  cells,  the  latter  immediately 
dissolved.  Now,  to  the  clear  centrifuged  fluid, 
which  as  we  have  -seen  would  not  dissolve  rabbit 
red  cells,  they  added  guinea-pig  red  cells  and  again 
some  normal  horse  serum  to  reactivate  the  mixture. 
The  guinea-pig  red  cells  all  dissolved.  This  proved 
conclusively  that  in  the  normal  goat  serum  there 
had  existed  two  specific  inter-bodies.  One,  for 
rabbit  red  cells,  had  been  tied  by  these  cells  and 
carried  down  with  them  in  centrifuging ;  the  other, 
specific  for  guinea-pig  red  cells,  had  remained  behind. 

Multiplicity  of  the  Active  Substances. --These 
investigators  were  able  to  prove  still  more  in  regard 
to  the  multiplicity  of  the  substances  in  normal 
serum  which  are  concerned  in  haemolysis.  They 
showed  that  beside  the  two  inter-bodies  just  men- 
tioned there  existed  in  goat  serum  two  specific 
complements,  one  for  each  inter-body,  and  they 
were  able  by  means  of  Pukall  filters  to  separate 
these  two.  In  this  filtration  the  complement  fit- 
ting the  inter-body  for  rabbit  blood  remained 
behind  for  the  greater  part,  while  that  fitting 
the  inter-body  for  guinea-pig  blood  mostly  passed 
through. 

Whereas  then,  according  to  Buchner,  only  one 
substance,  the  alexin,  is  concerned  in  the  haemo- 
lytic  action  of  this  normal  goat  serum  these  experi- 


?6  IMMUNE  SERA      - 

ments  of  Ehrlich  and  Morgenroth  show  us  four 
substances,  viz.,  two  inter-bodies  and  two  comple- 
ments. This  at  once  makes  clear  the  opposing 
views  of  these  authorities.  But  the  number  of 
active  substances  in  normal  serum  is  still  greater, 
for  in  the  experiments  of  the  last-named  authors 
it  often  happens  that  a  specific  inter-body  shows 
itself  to  be  made  up  of  several  inter-bodies,  all,  to 
be  sure,  fitting  the  same  specific  red  cell,  but  dif- 
fering from  each  other  by  their  behavior  toward 
different  complements.  Ehrlich,  therefore,  regards 
the  substances  concerned  in  haemolysis  which  occur 
in  normal  serum  to  be  of  great  number  and  variety. 
Buchner  and  Bordet,  on  the  other  hand,  assume 
that  only  one  substance  is  concerned. 

Difference  between  a  Normal  and  a  Specific 
Immune  Serum.  —  Practical  Application.  —  Return- 
ing now  to  the  question  of  the  difference  between  a 
specific  immune  serum  and  a  normal  one,  we  find 
this  to  be  as  follows :  Normal  serum  contains  a  great 
variety  of  inter-bodies,  in  very  small  amounts,  and  a 
considerable  amount  of  complements.  In  immune 
serum,  on  the  other  hand,  the  amount  of  a  specific 
inter-body,  the  one  which  fits  the  haptophore  group 
of  a  certain  cell,  is  enormously  increased.  This 
specifically  increased  inter-body,  it  will  be  remem- 
bered, is  called  the  immune  body.  The  comple- 
ment, as  shown  by  v.  Dungern,  Bordet,  Ehrlich, 
and  Morgenroth  and  Wassermann,  is  in  no  way 


BACTERIOLYSINS  AND  H&MOLYS1NS          77 

increased  by  the  immunizing  process.  The  increase 
affects  solely  the  immune  body.  It  is  therefore 
possible  to  have  a  serum  which  contains  more 
immune  body  than  complement  to  satisfy  it,  and  if 
we  withdraw  such  a  serum  from  an  animal  we  shall 
find  that  it  contains  some  free  immune  body.  This 
serum  can  only  then  exert  its  full  power  when  the 
full  amount  of  complement  is  present,  i.e.,  when 
some  normal  serum  is  added.  If  we  treat  a  rabbit 
with  the  red  cells  of  an  ox,  as  v.  Dungern  did,  we 
shall  obtain  a  serum  which  is  hn^mplytic  for  ox 
blood.  Of  this  freshly  drawn  serum  0.05  c.c.  suf- 
fice to  dissolve  5.0  c.c.  of  a  5%  mixture  of  ox 
blood.  If  now  we  add  to  this  haemolytic  serum 
a  little  normal  rabbit  serum,  we  shall  find  that 
only  one-tenth  of  the  amount  of  serum  is  required; 
i.e.,  only  0.005  c-c-  to  dissolve  the  same  quantity 
of  ox  blood.  This  means  that  through  the  addi- 
tion of  the  rabbit  serum,  which,  of  course,  is  not 
haemolytic  for  ox  blood,  a  sufficient  amount  of 
complement  was  added  to  enable  all  -the  immune 
body  of  the  specific  serum  to  act.  This  specifically 
increased  power  of  the  immune  serum  to  act  on 
certain  definite  cells  depends  on  the  fact  that  the 
immune  body  resulting  from  the  immunizing 
process  concentrates  the  action  of  the  comple- 
ment scattered  through,  the  serum,  on  cells  for 
which  it  has  definite  affinities.  If  2  c.c.  of  normal 
guinea-pig  serum  are  able  to  dissolve,  we  will  say. 


78  IMMUNE  SERA 

5  c.c.  of  a  5%  defibrinated  rabbit-blood  mixture, 
and  if  we  find  that  after  the  immunizing  process 
0.05  c.c.  of  the  guinea-pig  serum  suffice  to  dissolve 
the  same  amount  of  rabbit  blood,  we  conclude 
that  through  this  process  the  inter-body,  i.e.  the 
immune  body,  has  been  increased  forty  times.  We 
know  that  the  complement  has  not  been  increased, 
but  this  is  now  able  to  act  by  means  of  forty  times 
increased  combining  facilities.  This  increase,  how- 
ever, is  exclusively  for  rabbit-blood  cells.  In  a 
bactericidal  immune  serum  this  specific  increase  is 
sometimes  as  much  as  100,000  times  that  of  normal 
serum. 

The  practical  idea  to  be  gained  from  this  for 
the  therapy  of  infectious  diseases  is  this:  that 
with  the  injection  of  an  immune  serum  we  supply 
only  one  of  the  necessary  constituents  to  kill 
and  dissolve  the  bacteria,  and  that  is  the  immune 
body. 

We  do  not,  however,  supply  the  second,  i.e.  the 
complement,  "for  this  we  have  seen  is  not  increased 
by  the  immunizing  process.  As  matters  stand, 
then,  the  use  of  a  specific  immune  serum  for 
therapeutic  purposes  assumes  that  the  complement 
which  fits  exactly  to  the  immune  body  and  which  is 
essential  for  the  latter 's  action  will  be  found  in  the 
organism  to  be  treated.  Since  in  certain  infec- 
tious diseases  the  required  complement  is  present 
in  too  small  amounts  in  the  organism,  Wassermann 


BACTERIOLYSINS  AND  HALMOLYSINS          79 

suggested  that  the  curative  power  of  many  bacteri- 
cidal sera  might  be  increased  by  the  simultaneous 
injection  of  the  sera  of  certain  normal  animals  in 
order  thus  to  gain  an  increased  amount  of  comple- 
ment; but  we  shall  soon  see  that  this  procedure, 
while  of  great  value  in  animal  experiments,  presents 
certain  difficulties. 

Nature  of  the  Immune  Body  —  Partial  Immune 
Bodies  of  Ehrlich.  -  -  Turning  now  to  a  closer  study  of 
the  nature  of  the  immune  body,  we  again  find  a  dif- 
ference of  opinion.  Whereas  Bordet,  Metchnikoff, 
and  Besredka  assume  each  immune  body  to  be  a 
single  definite  substance,  Ehrlich  and  Morgenroth 
as  a  result  of  their  experiments  hold  to  a  plurality 
of  bodies. 

These  authors  say  that  each  immune  body 
is  built  up  of  a  number  of  partial-immune  bodies, 
a  point  to  which  we  have  already  alluded.  In 
support  of  this  view  they  offer  the  following  ex- 
periment. On  immunizing  a  rabbit  with  ox  blood, 
they  obtained  a  serum  haemolytic  not  only  for 
ox  blood  but  also  for  goat  blood;  on  immunizing 
a  rabbit  with  goat  blood  they  obtained  a  serum 
haemolytic  for  goat  blood  and  ox  blood.1 

The  conditions  present  can  be  readily  under- 
stood by  reference  to  Fig.  7,  which  represents 
schematically  three  portions  of  the  combining  groups 

1  We  have  already  called  attention  to  these  exceptions  to  the 
rule  of  specific  action. 


8o 


IMMUNE  SERA 


of  the  blood  cells.  Of  these  a  is  present  only  in  the 
ox-blood  cells,  ^  only  in  the  goat-blood  cells,  and  ft 
in  both.  If  a  rabbit  is  injected  with  ox  blood,  the 
immune  bodies  corresponding  to  groups  a  and  ft 
will  be  formed.  On  subjecting  such  a  serum  to 
absorption  with  ox-blood  cells  we  shall  find  that 
these,  by  means  of  their  a  and  ft  groups  will  be  able  to 
absorb  all  the  immune  bodies,  whereas  goat-blood 
cells  will  in  a  similar  test  absorb  only  the  immune 


FIG.  7 


body  of  portion   ft,   leaving   the   immune   body   of 
portion  a  in  solution. 

According  to  Ehrlich's  theory,  then,  the  red  cells 
of  the  ox  possess  certain  receptors  which  are  identi- 
cal with  receptors  possessed  by  the  goat  red  cells. 
From  this  it  follows  that  in  a  single  red  cell  there 
are  several  or  many  groups  each  of  which  is  able, 
when  it  finds  a  fitting  receptor,  to  take  hold  of  a 


BACTERIOLYSIS  AND  H&MOLYSINS          8 1 

single  immune  body.  Ehrlich  and  Morgenroth, 
therefore,  claim  that  the  immune  body  of  a  haemo- 
lytic  serum  is  composed  of  the  sum  of  the  partial 
immune  bodies  which  correspond  to  the  individual 
receptors  used  to  excite  the  immunity.  It  may  be 
assumed,  then,  that  not  all  of  the  combining  groups 
of  a  cell,  be  this  a  blood  cell  or  a  bacterium,  will 
find  fitting  receptors  in  every  animal  organism, 
and  that  therefore  not  all  the  possible  partial  im- 
mune bodies  will  be  equally  developed.  In  one 
animal  there  may  be  receptors  which  are  not  pres- 
ent in  another,  and  in  this  way  there  might  be  a  dif- 
ferent variety  of  partial  immune  bodies  in  the  two 
animals.  This  would  lead  to  the  possibility  of  the 
occurrence  of  immune  bodies,  for  the  same  species 
of  blood  cell  or  bacterium,  differing  from  each  other 
in  the  partial  immune  bodies  composing  them, 
according  to  the  variety  of  animals  used  in  prepar- 
ing the  serum. 

Metchnikoffs  Views  —  Practical  Importance  of 
th<:  Point,  -  -  This  view  is  directly  opposed  to  that  of 
M  etchnikoff  and  Besredka,  who  believe  that  a  cer- 
tc  in  immune  body,  e.g.  one  specific  for  ox  blood, 
is  always  the  same  no  matter  from  what  animal  it 
is  derived.  The  point  is  not  merely  theoretical, 
b  it  under  certain  circumstances  of  great  practical 
importance.  If  we  believe,  as  Ehrlich  does,  that 
the  immune  body  differs  according  to  the  species  of 
animal  from  which  it  is  derived,  i.e.,  that  it  is  made 


82  IMMUNE  SERA 

up  of  different  partial-immune  bodies,  then  we  must 
admit  that  we  have  better  chances  for  finding  fit- 
ting complements  if  we  make  use  of  immune  bodies 
derived  from  a  variety  of  animals.  We  would,  for 
instance,  be  likely  to  achieve  better  results  in  treat- 
ing a  typhoid  patient  with  a  mixture  of  specific 
bactericidal  typhoid  sera  derived  from  a  variety  of 
animals  than  if  we  used  a  serum  derived  only  from 
a  horse.  For  in  such  a  mixture  of  immune  bodies 
the  variety  of  partial-immune  bodies  must  be  very 
great  and  the  chances  that  the  complements  of  the 
human  body  will  find  fitting  immune  bodies,  and  so 
lead  to  the  destruction  of  the  typhoid  bacilli,  are 
greatly  increased.  Ehrlich  and  his  pupils  have 
actually  proposed  such  a  procedure  in  the  use  of 
bactericidal  sera  for  therapeutic  purposes.1 

Support  for  Ehrlich' s  View.  —  Besides  the  above 
experiments  we  possess  others  which  support  the 
theory  that  the  immune  body  is  not  a  simple  but 
a  compound  substance,  v.  Dungern  had  alre&dy 
shown  that  following  the  treatment  of  an  animal 
with  ciliated  epithelium  from  the  trachea  of  an  c  x, 
there  were  developed  immune  bodies  which  act  3d 
not  only  on  the  ciliated  epithelium  but  also  on  tie 
red  cells  of  oxen.  We  must  assume,  therefore,  th  it 

1  Reasoning  along  similar  lines,  namely,  that  the  hurr  in 
complement  must  fit  the  immune  body  of  the  therapeutic 
serum,  Ehrlich  has  also  proposed  that  these  bactericidal  s.;ra 
be  derived  from  animals  very  closely  related  to  man,  e  g., 
apes,  etc. 


BACTERIOLYSINS  AND  HMMOLYSINS  83 

the  ciliated  epithelium  and  the  red  cells  of  the  ox 
possess  common  receptors.  Analogous  to  this  is 
the  action  of  the  immune  body  resulting  from  the 
injection  of  spermatozoa,  as  was  pointed  out  by 
Metchnikoff  and  Moxter. 

We  see,  then,  that  the  specific  action  of  immune 
bodies  is  not  so  limited  as  to  apply  only  to  the  cells 
used  in  the  immunizing  process,  but  extends  to 
other  cells  which  have  receptors  in  common  with 
these.1 

Coming  now  to  the  question  as  to  what  part  of 
the  cell  it  is  which  excites  the  production  of  the 
haemolytic  immune  body,  we  find  this,  according  to 
v.  Dungern,  to  be  the  stroma  of  the  red  cells.  If 
this  be  so,  it  must  be  the  stroma  which  combines 
with  the  immune  body.  Nolf,  however,  claims 
that  the  cell  contents  are  factors  in  the  production 
of  the  immune  body.  So  far  as  concerns  the  site 
in  the  organism  where  the  substances  used  in  immu- 
nizing find  their  receptors,  this  is  not  known  for 
the  hsemolytic  immune  body. 

For  the  bactericidal  immune  bodies  of  cholera, 
and  typhoid  the  researches  of  Pfeiffer,  Marx,  and 
others  show  that  the  chief  site  of  production  is  in 

1  The  same  holds  good  for  the  agglutinins  and  the  pre- 
cipitins  still  to  be  studied.  In  these  the  action  extends  also 
to  closely  related  cells  and  bacteria,  or  in  the  case  of  the  precipi- 
tins  to  closely  related  albumins,  as  these  possess  a  number  of 
receptors  which  are  common  to  them  and  to  the  cells  or  sub- 
stances used  for  immunizing. 


84  IMMUNE  SERA 

the  bone-marrow,  spleen,  and  lymph  bodies.  Was- 
sermann's  experiments  on  local  immunity  indicate 
that  the  site  of  infection  determines  largely  the  site 
of  the  development  of  the  immune  bodies. 

Antihaemolysins :  their  Nature  —  Anti-comple- 
ment or  Anti-immune  Body  ?  —  A  further  step  in  the 
study  of  hsemolysins  is  one  discovered  independ- 
ently by  Ehrlich  and  Morgenroth  on  the  one  hand, 
and  Bordet  on  the  other.  These  authors  succeeded 
in  producing  an  antih&molysin.  The  procedure  is 
closely  related  to  the  results  gained  by  immuniza- 
tion against  bacterial  poisons.  A  specific  haemoly- 
sin,  one,  for  example,  specific  for  rabbit  blood, 
derived  by  treating  a  guinea  pig  with  rabbit  red 
cells,  is  highly  toxic  to  rabbits.  Injected  into  the 
animals  intravenously  in  doses  of  5  c.c.  it  kills  the 
animals  acutely,  causing  intra  vitam  a  solution  of 
the  red  cells.  Such  a  hsemolytic  serum,  then,  acts 
the  same  as  a  bacterial  poison,  and  it  is  possible  to 
immunize  against  this  just  as  well  as  against  a  bac- 
terial poison.  For  example,  to  keep  to  our  illustra- 
tion, rabbits  are  injected  first  with  very  small  doses 
of  this  specific  hagmolytic  serum.  The  dose  is 
gradually  increased  until  it  is  found  that  the  animal 
tolerates  amounts  that  would  be  absolutely  fatal  to 
animals  not  so  treated.  If  some  of  the  serum  of 
this  animal  is  now  abstracted  and  added  to  the 
specific  haemolytic  serum,  it  is  found  that  the  power 
of  the  latter  will  be  inhibited.  This  shows  that  an 


BACTERIOLYSINS  AND  H^MOLYSINS          85 

antih&molysin  has  been  formed.  As  we  know  that 
the  action  of  the  haemolysin  depends  on  the  com- 
bined action  of  two  substances,  the  immune  body 
and  the  complement,  the  question  arises  to  which 
of  these  two  the  antihaemolysin  is  related.  Is  it 
an  anti-immune  body  or  an  anti-complement?  A 
study  of  this  question  shows  that  both  these  sub- 
stances are  apparently  present.  In  the  serum  of  the 
rabbit  treated  with  specific  haemolysin,  both  an  anti- 
immune  body  and  an  anti-complement  have  been 
found.  For  the  details  of  the  experiments  of 
Ehrlich  and  Morgenroth  and  of  Besredka,  which  dem- 
onstrated this,  I  must  refer  to  the  original  articles. 
The  first-named  authors  were  further  able  to  show 
that  the  action  of  the  anti-complement  depended 
on  a  haptophore  group  which  it  possessed,  enabling 
it  to  combine  with  the  haptophore  group  of  the 
complement,  thus  satisfying  this  and  hindering  its 
combination  with  the  complementophile  group  of 
the  immune  body. 

Anti-complement.  —  Since  the  complements  are 
constituents  of  normal  serum,  it  should  be  possible 
to  produce  anti-complements  by  injecting  animals 
merely  with  normal  serum;  and  they  can,  in  fact, 
be  so  produced.  If  rabbits  are  treated  by  inject- 
ing them  several  times  with  normal  guinea  pig 
serum,  a  serum  may  be  obtained  from  these  rabbits 
which  contains  anti-complements  against  the  com- 
plements of  normal  guinea-pig  serum.  A  serum 


86  IMMUNE  SERA 

obtained  in  this  way  of  course  contains  only  one  of 
the  antihaemolytic  bodies,  the  anticomplement, 
and  not  the  antiimmune  body.  This  is  because 
normal  serum  is  too  poor  in  immune  body  (inter- 
body)  to  excite  the  production  of  any  antiimmune 
body. 

If  to  a  hasmolytic  serum  derived  from  guinea  pigs 
we  add  an  anticomplement  serum  derived,  as  just 
stated,  from  rabbits,  and  containing  an  anticom- 


COMPLEMENT 
COMPLEMENT       ^^  ^^ 

ANTICOMPLEMENT 


IMMUNE    BODY    _  _  BQDY 


CELL    /  *!%/  CELL 


(After  Levaditi.) 


plement  specific  for  guinea-pig  complement,  the 
hgemolytic  action  of  the  former  will  be  inhibited,  for 
the  reason  that  the  complement  necessary  for  the 
hsemolysis  to  take  place  has  been  bound  by  the 
anticomplement.  (See  Fig.  8.)  One  must,  how- 
ever, observe  the  precaution  to  heat  the  anticom- 
plement serum  of  the  rabbit  to  55°C.  before  so 
mixing  it,  in  order  to  destroy  the  complement  which 
it  contains  and  which  would  otherwise  reactivate  the 
guinea-pig  immune  body. 


BACTERIOLYSINS  AND  H^EMOLYSINS          87 

From  the  foregoing  we  see  that  either  anti- 
immune  body  alone,  or  anticomplement  alone,  is 
able  to  inhibit  the  hsemolytic  action.  Haemoly- 
sis cannot  take  place  when  either  of  the  two 
necessary  factors  is  bound  and  prevented  from 
acting.1 

The  anticomplements  are  specific  bodies,  i.e.,  an 
anticomplement  combines  only  with  its  specific 
complement.  Thus  an  anticomplement  serum 
derived  from  rabbits  by  treatment  with  guinea- 
pig  serum  combines  only  with  the  complement  of 
normal  guinea-pig  serum,  not,  however,  with  the 
complements  of  other  animals.  Exceptions  to  this 
are  those  cases  in  which  the  complement  of  the 
other  species  possess  receptors  identical  with  those 
of  the  first. 

In  order  that  a  normal  serum  of  species  A, 
injected  into  species  B,  produce  anticomplements 
there,  the  side-chain  theory  demands  that  the  com- 
plements of  A  find  fitting  receptors  in  species  B. 
According  to  Ehrlich,  however,  normal  serum  con- 
tains many  different  complements  and  not  merely  a 
single  one.  Under  the  circumstances,  it  is  easily 
possible  that  only  a  few  of  the  complements  in  the 

1  By  treating  animals  with  normal  sera  of  certain  other 
species,  it  is  possible  to  produce  not  only  anti-complements, 
but  also  specific  anti-bodies  against  certain  other  constituents 
of  normal  serum.  These  are,  for  example,  anti-agglutinins, 
which  inhibit  the  action  of  the  haemagglutinins  of  normal  serum, 
and  anti-precipitins,  which  we  shall  discuss  later. 


88  IMMUNE  SERA 

serum  of  A  find  fitting  receptors  in  species  B.  We 
shall  then  obtain  an  anticomplement  serum  which 
inhibits  the  action  of  some,  but  not  of  all  the  com- 
plements of  species  A.  Thus  it  might  inhibit  the 
action  of  a  complement  fitting  to  a  certain  bacteri- 
cidal immune  body  and  not  of  one  contained  in  the 
same  serum  which  fitted  a  certain  hasmolytic  im- 
mune body,  etc. 

Auto-anticomplements .  — A  question  of  great  prac- 
tical importance  now  arises.  Is  it  possible  under 
certain  conditions  for  an  organism  to  manufac- 
ture within  itself  anticomplements  against  its 
own  complements,  i.e.,  auto -anticomplements  ?  The 
complements,  owing  to  their  ferment-like  digestive 
power,  must  play  an  important  role  in  the  living 
organism;  for  this  concerns  itself  not  only  with  the 
destruction  of  bacteria,  etc.,  an  important  factor  in 
the  natural  immunity  against  diseases,  but  also, 
according  to  Ehrlich,  Buchner,  and  Wassermann, 
with  the  solution  and  digestion  of  all  kinds  of  foreign 
albuminous  bodies  which  enter  the  organism.  Any 
inhibition  of  this  important  function  would  there- 
fore be  followed  by  severe  disturbances,  particu- 
larly, however,  by  a  decreased  resistance  against 
infectious  diseases.  Wassermann  succeeded  in  dem- 
onstrating that  animals  injected  with  anti -comple- 
ments to  tie  up  their  complements  were  much  less 
resistant  to  certain  infectious  diseases. 

The  spontaneous    development   in   an    animal    of 


BACTERIOLYSINS  AND  HJLMOLYSINS          89 

auto-anticomplement,  i.e.,  substances  developed 
within  the  organism  against  its  own  complements, 
has  not  yet  been  demonstrated.  Ehrlich  and  Mor- 
genroth  were  able  to  excite  the  production  of  an 
auto-anticomplement  in  a  rabbit  by  treating  the 
animal  in  a  certain  way.  Ordinarily,  normal  rab- 
bit serum  is  slightly  solvent  for  guinea-pig  blood. 
If  the  rabbits  are  treated  with  goat  serum,  the  rab- 
bit serum  loses  this  solvent  power  for  guinea-pig 
red  cells.  Even  if  fresh,  normal  rabbit  serum  is 
now  added,  haemolysis  does  not  take  place,  although 
we  know  that  this  fresh  serum  is  haemolytic.  This 
shows  that  in  the  serum  of  the  rabbit  treated  with 
goat  blood,  an  anticomplement  has  been  formed 
which  combines  with  the  complement  of  normal 
rabbit  blood,  for  it  was  able  to  inhibit  the  action 
of  the  complement  of  the  normal,  freshly  added 
rabbit  serum.  In  the  rabbit's  body,  then,  as  a 
result  of  this  procedure,  an  anticomplement  has 
been  formed  against  the  complement  of  its  own 
serum,  a  true  auto-anticomplement. 

Now,  according  to  the  side-chain  theory,  there 
are  no  receptors  in  an  organism  for  the  complements 
of  the  same  organism.  The  formation  of  these 
auto-anticomplements,  according  to  Ehrlich,  can 
only  be  explained  by  assuming  that  in  normal  goat 
serum  there  are  present  complements  which  are 
almost  identical  with  those  of  the  rabbit  serum, 
but  which  differ  from  them  in  that  they  find  recep- 


QO  IMMUNE  SERA 

tors  in  the  rabbit  serum  whose  haptophore  group 
fits  to  their  own. 

Fluctuations  in  the  Amount  of  the  Active  Sub- 
stances in  Serum.  —  As  already  said,  we  have  thus 
far  been  unable  to  show  that  the  complements  of  an 
organism  are  decreased  through  the  action  of  spon- 
taneously formed  anticomplements.  We  have, 
however,  come  to  know  certain  conditions  under 
which  there  may  be  a  decrease  of  certain  comple- 
ments in  normal  serum.  Ehrlich  and  Morgenroth 
showed  that  in  rabbits  poisoned  with  phosphorus 
and  in  whom,  therefore,  the  liver  was  badly  damaged, 
the  serum  on  the  second  day  (the  height  of  the 
disease)  had  lost  its  power  to  dissolve  guinea-pig 
blood,  and  that  this  was  due  to  a  disappeareance  of 
the  complement.  Metchnikoff  also  reported  that  in 
an  animal  suffering  from  a  continuing  suppurating, 
process,  the  complement  had  fallen  considerably  in 
amount.  Especially  interesting  are  the  experi- 
ments of  v.  Dungern,  who  showed  that  animal  cells, 
hence  emulsions  of  fresh  organs,  are  able  to  attract 
and  combine  with  complements. 

Even  more  important  than  the  question  of  a 
decrease  in  complements,  or  an  inhibition  of  their 
action,  is  that  of  the  possibility  to  artificially  in- 
crease them.  A  number  of  authors,  among  them 
Nolf  and  Miiller,  have  answered  this  question  in  the 
affirmative.  They  believe  they  have  noticed  such 
an  increase  following  the  injection  of  an  animal  with 


BACTER1OLYSINS  AND  H^EMOLY SINS  Ql 

all  sorts  of  substances,  such  as  normal  serum  of 
another  animal,  sterile  bouillon,  etc.  v.  Dungern, 
Wassermann  and  others,  have  not  been  able  to  con- 
vince themselves  of  the  possibility  of  such  a  definite 
increase.  Wassermann  tried  to  excite  the  increased 
production  of  complement  by  injecting  guinea  pigs 
for  some  time  with  anticomplement.  This  being 
the  opposite  of  the  complement,  he  hoped  to  be 
able  by  immunizing  to  excite  an  increase  of  the 
complements.  In  this  he  was  unsuccessful,  though 
of  course  it  may  be  possible  with  another  species  of 
animal. 

Despite  all  this,  we  must  believe  that  the  amount 
of  complement,  as  well  as  the  amount  of  other  active 
substances  of  the  blood,  inter-bodies,  agglutinins, 
antitoxins,  ferments,  antiferments,  etc.,  is  subject 
to  great  fluctuations  even  in  the  same  individual, 
a  constant  change  going  on  within  the  organism. 
Ehrlich,  in  particular,  has  pointed  out  these  indi- 
vidual and  periodic  variations  and  has  insisted  on 
their  importance.  Very  likely,  under  circumstances 
of  which  we  now  know  very  little,  these  substances 
are. at  certain  times  produced  in  greater  amounts, 
at  other  times  in  lesser;  sometimes  they  may  be 
absent  entirely  in  an  individual  in  whom  they  were 
previously  present.  For  example,  the  serum  of  a 
dog  will  at  times  dissolve  the  red  cells  of  cats,  rab- 
bits, and  guinea  pigs,  at  other  times  not.  Further- 
more, the  serum  of  one  and  the  same  animal  may 


92  IMMUNE   SERA 

possess  specific  haemolytic  properties  for  certain 
cells,  and  later  on  may  lose  this  property  entirely. 
In  human  serum  these  same  individual  and  periodic 
variations  may  be  demonstrated,  as  Wassermann 
was  able  to  prove  experimentally.  However,  the 
circumstances  on  which  these  variations  depend  are 
as  yet  entirely  unknown  to  us.  Possibly  we  are 
dealing  here  with  subtle  pathological  changes. 

Source  of  the  Complements  —  Leucocytes  as  a 
Source  —  Other  Sources.  —  Where  do  the  comple- 
ments or  alexins  originate?  This  question  has  been 
studied  particularly  by  MetchnikofI  and  by  Buch- 
ner;  also  by  Bail,  Hahn,  Schattenfroh,  and  others. 
These  investigators  believe  that  the  leucocytes  are 
the  source  of  the  complements  or  alexins.  There 
is,  however,  this  difference  between  the  views 
of  Metchnikoff  and  Buchner:  whereas  Buchner 
believes  the  alexins  to  be  true  secretory  products, 
Metchnikoff  believes  that  they  originate  on  the 
breaking  up  of  the  leucocytes,  i.e.,  that  they  are  de- 
composition products.  Metchnikoff  bases  his  belief 
chiefly  on  the  work  of  his  pupil,  Gengou,  who  showed 
that  although  the  serum  was  rich  in  alexin  (i.e.,  com- 
plement) the  plasma  contained  none  at  all. 

Other  authors,  as  Pfeiffer  and  Moxter,  as  a  result 
of  their  experiments,  are  not  willing  to  assume  the 
existence  of  any  relationship  between  the  alexins 
and  the  leucocytes.  Gruber  as  well  as  Schatten- 
froh are  ready  to  believe  the  leucocytes  to  be  the 


BACTERIOLYSINS  AND   H.EMOLYSINS  93 

source  of  an  alexin,  but  claim  that  this  is  different 
from  that  found  in  serum.  Wassermann  believes 
that  the  leucocytes  are  a  source  of  complements 
(alexins),  for  he  succeeded  in  producing  anticom- 
plement  by  means  of  injections  of  pure  leucocytes 
which  had  been  washed  free  from  all  traces  of  serum, 
and  which  had  been  obtained  by  injections  of  aleu- 
ronat.  In  view  of  the  plurality  of  the  comple- 
ments, Wassermann  expressed  the  view  that  the 
leucocytes  are  probably  one  source,  but  not  the 
only  one,  for  the  complements  of  the  serum.  Land- 
steiner  and  Donath  have  confirmed  this  experi- 
mentally. They  succeeded  in  producing  anticom- 
plement  by  the  injection,  not  only  of  leucocytes, 
but  of  other  animal  cells.  Furthermore,  the  experi- 
ments of  Ehrlich  and  Morgenroth  already  mentioned, 
in  which  the  complements  disappeared  after  the 
destruction  of  the  liver  function,  show  that  the  liver 
cells  are  concerned  in  the  formation  of  complements. 
Structure  of  Complements  —  Haptophore  and  Zy- 
motoxic  Groups  —  Complementoids.  -  -  The  structure 
of  the  complement  has  been  studied  particularly  by 
Ehrlich  and  Morgenroth,  and  by  P.  Miiller.  We 
have  seen  that  the  complements  lose  their  power 
when  heated  to  55°C.  If,  however,  we  inject  ani- 
mals with  a  normal  serum  that  has  previously  been 
heated  to  55°C.,  we  shall  still  excite  in  these  ani- 
mals the  production  of  anticomplements.  This 
shows  that  the  heating  has  not  destroyed  the  entire 


94  IMMUNE  SERA 

complement  body,  but  only  that  part  which  affects 
the  digesting,  solvent  action.  The  part  of  the 
complement  concerned  with  the  combination  with 
the  inter-body  or  immune  body,  in  other  words, 
that  part  called  by  Ehrlich  the  haptophore  group, 
must  have  remained  intact.  It  is  clear,  therefore, 
that  anticomplements  can  only  be  formed  when 
there  remain  in  the  complements  haptophore  groups 

zymotoxic  group 
COMPLEMENT 
haptophore  group 

IMMUNE   BODY 
FlG.  9. 

that  fit  certain  receptors  in  the  organism  of  the 
animal  injected.  From  this  it  follows  that  the 
complements  consist  of  a  combining  haptophore 
group  which  withstands  heating  to  55°C.,  and 
another  more  fragile  group  which  possesses  the 
actual  solvent  properties,  and  which  Ehrlich  calls 
the  zymotoxic  group.  There  is  a  perfect  analogy  be- 
tween this  and  the  toxins  already  studied.  These, 
it  will  be  remembered,  consist  of  a  haptophore  and 


BACTERIOLYS1NS  AND  H&MOLYSINS          95 

a  toxophore  group.  And  just  as  those  toxins  which 
had  lost  their  toxophore  group  were  called  toxoids, 
so  Ehrlich  and  Morgenroth  purpose  to  call  com- 
plements which  have  lost  their  zymotoxic  group, 
complementoids . 

Isolysins  —  Autolysins  —  Anti-isolysins.  —  All  of 
the  preceding  studies  in  haemolysis  have  concerned 
themselves  with  the  results  obtained  by  injecting 
animals  of  one  species  with  blood  cells  of  another. 
Ehrlich  and  Morgenroth  now  sought  to  discover 
what  the  results  would  be  if  they  injected  an  animal 
with  blood  cells  of  its  own  species.  They  injected 
goats  with  goat  blood,  and  found  that  when  the 
amount  injected  at  one  time  was  large  the  serum 
of  the  goat  injected  acquired  hasmolytic  properties 
for  the  blood  of  many  other  goats,  but  not  for  all. 
The  substances  thus  formed  the  authors  called 
isolysins.  These,  then,  are  substances  which  will 
dissolve  the  blood  of  other  individuals  of  the  same 
species.  Substances  which  dissolve  the  blood  cells 
of  the  same  individual  are  called  autolysins.  But 
autolysins  have  so  far  been  demonstrated  experi- 
mentally only  once  (by  Ehrlich  and  Morgenroth). 
If  one  tests  the  properties  of  an  isolysin  of  a  goat  on 
the  blood  of  a  great  many  other  goats,  it  will  be 
found  that  this  will  be  strongly  solvent  for  the 
blood  of  some,  slightly  for  the  blood  of  others,  and 
not  at  all  for  still  others. 

By  using  a  blood  that  was  readily  dissolved  by 


96  IMMUNE  SERA 

the  isolysin,  and  proceeding  in  the  same  series  of 
experiments  which  we  have  already  studied  under 
haemolysis,  Ehrlich  and  Morgenroth  showed  that 
the  isolysins,  like  the  haemolysins,  consist  of  an 
immune  body  and  a  complement  of  the  normal 
serum.  The  experiments  undertaken  by  these 
authors  were  made  on  thirteen  goats,  and  the  sur- 
prising fact  developed  that  the  thirteen  resulting 
isolysins  were  all  different.  For  example,  the  iso- 
haemolytic  serum  of  one  goat  dissolved  the  red  cells 
of  goats  A  and  B;  that  of  a  second  goat  those  of 
C  and  D ;  of  a  third  those  of  A  and  D,  but  not  of  (7, 
and  so  on.  If  now  they  produced  antiisolysins  by 
injecting  animals  with  these  isolysins,  they  found 
that  these  antiisolysins  were  specific;  i.e.,  the  anti- 
isolysin  of  A  would  inhibit  the  action  only  of  iso- 
lysin of  A,  but  not  of  C,  etc.  These  results  are  of 
the  highest  clinical  interest,  for  they  show  a  differ- 
ence in  similar  cells  of  the  same  species,  something 
that  had  never  before  been  suspected.  In  the 
above,  the  blood  cells  of  species  A  must  have  a  dif- 
ferent biological  constitution  than  those  of  species 
C,  etc. 

The  fact  that  after  injections  of  large  amounts  of 
cells  of  the  same  species  isolysins  develop,  but  that 
autolysins  are  almost  never  formed,  caused  Ehr- 
lich and  Morgenroth  to  assume  that  the  body  pos- 
sesses distinct  regulating  functions  which  naturally 
prevent  the  formation  of  the  highly  destructive 


BACTER1OLYSINS  AND  H&MOLYSINS  97 

autolytic  substance.  It  is  obvious  that  if  there 
were  no  such  regulating  facilities,  the  absorption  of 
large  bloody  effusions  and  hemorrhages  might  lead 
to  the  formation  by  the  organism  of  autolysins 
against  its  own  blood  cells.  Gengou,  a  pupil  of 
Metchnikoff,  believes  to  have  shown  experimen- 
tally that  the  destructive  action  of  these  auto- 
lysins is  hindered  by  the  simultaneous  production 
of  an  auto-antiimmune  body  which  immediately 
inhibits  their  action. 

In  order  that  isolysins  may  be  formed,  it  seems 
necessary  to  overwhelm  the  organism  once  or  sev- 
eral times  with  large  amounts  of  cells  or  cell  prod- 
ucts of  the  same  species ;  to  produce,  as  Ehrlich  says, 
an  ictus  immunisatorius.  Wassermann  tried,  by 
using  various  blood  poisons,  such  as  hasmolytic  sera, 
toluylenediamine,  etc.,  for  a  continued  length  of 
time,  to  cause  the  formation  of  these  isolysins,  but 
without  success,  although  in  these  experiments 
each  injection  was  followed  by  an  appreciable 
destruction  of  red  cells  and  absorption  of  their 
decomposition  products.  The  gradual  and  even 
repeated  absorption  of  not  too  large  quantities  of 
decomposed  red  cells  does  not  therefore  lead  to  the 
formation  of  isolysins;  but,  as  already  said,  a  sudden 
overwhelming  of  the  organism  by  large  amounts 
of  the  cells  or  their  products  is  necessary. 

Deflection  of  Complement.  —  In  the  use  of  the 
antitoxic  sera,  experience  has  shown  that  the  em- 


98  IMMUNE  SERA 

ployment  of  a  large  dose  is  of  paramount  importance. 
So  far  as  the  antitoxic  action  is  concerned  *  one 
cannot  do  harm  by  giving  a  large  excess.  Con- 
cerning the  action  of  bactericidal  sera,  however, 
the  literature  contains  a  number  of  examples  which 
indicate  that  here  an  excess  of  immune  serum  is 
occasionally  injurious.  Perhaps  the  earliest  proto- 
col of  this  kind  is  that  published  by  Loffler  and 
Abel 2  on  their  experiments  with  bacillus  coli  and  a 
corresponding  immune  serum.  Out  of  nineteen 
guinea  pigs  which  had  been  inoculated  with  the 
same  amount  of  culture  and  had  received  varying 
amounts  of  immune  serum,  only  six  animals  were 
protected,  those  which  had  received  doses  of  0.25 
c.c,  to  0.02  c.c.  Eight  animals  with  larger  doses, 
as  well  as  five  with  smaller  doses  of  serum  died. 
Neisser  and  Wechsberg 3  encountered  the  same 
phenomenon  in  bactericidal  test-tube  experiments, 
and  concluded  as  a  result  of  their  experiments 
that  the  only  satisfactory  explanation  was  one  based 
on  the  views  of  Ehrlich  and  Morgenroth.  In  Fig. 
10,  A  II  represents  schematically  a  bacterium  a 
with  a  number  of  receptors;  for  there  are  many 
reasons  for  assuming  that  each  bacterium  possesses  a 

1  We  shall  discuss  the  rash  production,  or  "  serum  sickness," 
page  138. 

2  F.  Loffler  and  R«  Abel,  Centralblatt  Bacteriol.,   1896,  Vol. 
xix,  p.  51. 

8  M.    Neisser    and    F.    Wechsberg,    Munch,    rned.    Wochen- 
schrift.  1901.     No.  18. 


BACTERIOLYSINS  AND  H&MOLYSINS          99 


£=<? 


100  IMMUNE  SERA 

number  of  receptors  of  the  same  kind.  According 
to  the  side-chain  theory,  if  we  inject  this  bacterium 
into  an  animal  an  over-production  of  the  corres- 
ponding group  will  occur,  resulting  in  a  serum 
which  is  rich  in  body  b.  This  body  6,  however,  is 
not  able  by  itself  to  injure  the  bacteria,  and  a 
bacterium  all  of  whose  receptors  are  laden  with  b 
need  not  at  all  be  injured  in  its  vitality.  Body  b 
normally  possesses  a  peculiar  function,  namely, 
to  serve  as  a  coupling  member  or  link,  and  hence  it 
possesses  two  groups  (amboceptor).  A$  has  already 
been  discussed,  one  of  these  groups  fit  the  receptors 
of  the  bacterium  on  the  one  hand  and  the  com- 
plement on  the  other.  When,  therefore,  to  a  normal 
serum  which  contains  suitable  complement,  we  add 
equivalent  amounts  of  immune  serum,  the  con- 
dition pictured  in  A  I  will  result.  On  adding  the 
corresponding  bacterium  to  this  we  get  the  con- 
dition shown  in  A  II,  in  which  all  the  bacterial 
receptors  are  occupied  with  immune  bodies,  or 
more  accurately,  with  immune  bodies  which  on 
their  part  are  loaded  with  bacteriolytic  comple- 
ment c.  In  the  case  here  presented  let  us  say  that 
it  requires  the  occupation  of  all  of  the  receptors 
with  complemented  interbodies  to  cause  the  death 
of  the  bacterium. 

If  now  to  an  equivalent  mixture  of  comple- 
ment and  inter-body  we  add  an  excess  of  inter-body, 
it  will  be  possible  for  only  a  part  of  the  inter-body  to 


BACTERIOLYSINS  AND   HMMOLYSINS         IOI 

be  loaded  with  complement,  leaving  a  portion  of 
the  inter-body  uncomplemented.  On  adding  the 
corresponding  bacteria  a  number  of  conditions  may 
result;  the  affinity  of  the  inter-body  for  the  bac- 
terial receptor  may,  as  a  result  of  the  loading  with 
complement,  (i)  remain  unchanged,  (2)  it  may 
thereby  be  increased,  or  (3)  be  diminished. 

In  the  figure,  B  II  shows  the  condition  of  in- 
creased affinity.  Of  the  six  inter-bodies  only  those 
combine  with  the  bacterium  which  have  become 
laden  with  complement.  In  this  case,  therefore, 
the  excess  of  inter-bodies  will  have  no  influence  on 
the  bactericidal  effect.  The  condition  is  really  the 
same  as  A  II,  except  that  free  inter-body  is  also 
present. 

C  II  shows  the  condition  of  unchanged  affinity. 
In  this  case,  if  we  add  the  bacterium  to  the  mixture 
of  complement  and  excess  of  inter-body,  all  the 
receptors  of  the  bacterium  will,  to  be  sure,  be  occu- 
pied by  inter-bodies,  but  this  will  be  entirely  with- 
out regard  to  the  fact  that  these  inter-bodies  are  or 
are  not  loaded  with  complement.  It  may  there- 
fore happen  that  only  a  few  of  the  bacterial  receptors 
will  be  occupied  by  complemented  (i.e.,  active) 
inter-bodies,  while  the  rest  of  the  bacterial  receptors 
are  occupied  by  uncomplemented  (hence  inactive) 
inter-bodies.  As  already  stated,  however,  the 
vitality  of  such  a  bacterium  is  not  necessarily 
destroyed. 


102  IMMUNE  SERA 

D  II  represents  the  last  conceivable  case.  It  is 
assumed  that  the  "  completion  "  of  the  inter-body 
has  resulted  in  a  diminution  of  the  latter's  affinity 
for  the  bacterial  receptor.  In  this  case  primarily 
only  the  uncomplemented  inter-bodies  will  com- 
bine with  the  bacterial  receptors,  while  the  free 
fluid  will  contain  complemented  inter-bodies. 

In  cases  C  II  and  D  II,  therefore,  the  excess  of 
inter-body  exerts  a  deflecting  action  on  the  complement, 
thus  diminishing  the  end  results. 

It  is  difficult  to  say  to  what  extent  "  deflection  of 
complement "  really  occurs  in  the  experiments 
referred  to  above.  Recent  studies  by  Buxton x 
and  others  show  that  deflection  of  complement  will 
not  always  explain  the  phenomenon,  and  that  in 
these  instances  other  factors  must  be  responsible 
for  the  paradoxical  results. 

For  the  absorption  of  complement  commonly 
known  as  the  "  Bordet-Gengou,"  or  the  "  Gengou- 
Moreschi  "  phenomenon,  see  page  68.  To  avoid 
confusion  it  will  be  well  to  restrict  the  term  "  deflec- 
tion of  complement  "  to  the  phenomenon  described 
by  Neisser  and  Wechsberg. 

Deutsch's  Hsemolytic  Blood  Test.  —  Deutsch  2  in 
1900  suggested  the  use  of  artificial  haemolysins  in 
legal  medicine,  in  the  identification  of  bloods, 

1  Buxton;  Journal  Medical  Research,  Vol.  xiii,  1905. 
3  Deutsch,     Die     forensische      Serumdiagnose      des     Blutes, 
Centralblatt  Bacteriol.,   Vol.   xxix,    1901. 


B ACT ERIOLY SINS  AND  H&MOLYSINS         103 

both  fresh  and  dried.  He  found  that  a  powerful 
haemolytic  serum  dissolved  powdered  blood  com- 
pletely, the  latter  being  suspended  in  0.9%  salt 
solution.  Dried  blood  to  which  saline  is  added 
brings  the  haemoglobin  of  the  injured  corpuscles 
into  solution,  the  uninjured  corpuscles  do  not,  how- 
ever, dissolve  even  after  twenty-four  hours  at  37°  C. 
If  the  dried  blood  is  extracted  in  normal  rabbit 
serum,  more  haemoglobin  goes  into  solution  than 
with  saline,  when  the  proportion  added  is  1:2, 
whereas  the  normal  serum  acts  like  saline  when 
added  in  the  proportion  of  1:4.  When  two  samples 
of  the  same  dry  blood  are  brought  into  suspension 
in  normal  and  artificial  hsemolytic  serum,  respect- 
ively, a  little  phenol  or  toluol  being  added,  the  anti- 
serum  brings  about  complete  haemolysis  after  twenty- 
four  hours,  besides  leading  to  the  formation  of  a 
precipitum,  due  to  the  action  of  precipitins  formed 
in  the  blood-treated  animal  in  consequence  of  the 
serum  which  was  injected  together  with  the  cor- 
puscles. When  washed  corpuscles  alone  are 
injected  precipitins  are  not  formed.  In  view  of 
the  specificity  of  the  reactions  observed  with 
human  blood,  Deutsch  considers  that  the  method 
can  be  put  to  use  in  a  practical  way.  There 
can,  however,  be  no  question  but  that  the  pre- 
cipitins offer  many  advantages  over  the  haemoly- 
sins  for  such  purposes. 

For  other  biological  blood  tests  see  the  Wasser- 


104  IMMUNE  SERA 

mann-Uhlenhuth  precipitin  test,  page  112,  and  the 
recent  Neisser-Sachs  test,  page  70. 

Practical  Value  of  Injections  of  Bactericidal  Sera. 

-  The  use  of  sera  having  specific  protective 
properties  has  been  tried  practically  on  a  large 
scale  in  man  as  a  preventive  of  infection.  It  is 
difficult  to  estimate  just  what  value  these  injections 
have  had.  In  susceptible  animals,  injections  of 
some  of  the  very  virulent  bacteria,  as  pneumococci, 
streptococci,  typhoid  bacilli,  and  cholera  spirilla, 
can  be  robbed  of  all  danger  if  small  doses  of  their 
respective  serums  are  given  before  the  bacteria 
have  increased  to  any  great  extent  in  the  body. 
If  given  later  they  are  ineffective.  For  some 
bacteria,  such  as  tubercle  bacilli,  no  serum  has 
been  obtained  of  sufficient  power  to  surely  prevent 
infection.  Through  bactericidal  serums,  therefore, 
we  can  immunize  against  an  infection,  and  even 
stop  one  just  commencing;  but  as  yet  we  cannot 
cure  an  infection  which  is  already  fully  developed, 
though  even  here  there  is  reason  to  believe  that  we 
may  possibly  prevent  an  invasion  of  the  general 
system  from  a  diseased  organ,  as  by  the  pneumo- 
coccus  from  an  infected  lung  in  pneumonia.  On 
the  whole,  the  bactericidal  sera  have  not  given,  as 
observed  in  practice,  conclusive  evidence  of  great 
value  in  already  developed  disease. 

It  is  apparent  from  all  that  has  been  said  that  a 
deeper  insight  into  the  mechanism  of  the  bacteri- 


BACTERIOLYSINS  AND  H&MOLYS1NS         IO5 

cidal  sera  has  disclosed  many  difficulties  to  be 
overcome  before  we  can  hope  for  much  in  a  practi- 
cal way.  Thus  we  have  as  yet  found  no  method  of 
increasing  the  complements,  and  these  are  apparently 
highly  important  in  destroying  the  invading  bacteria. 
Nor  have  we  any  way  to  determine  the  proper 
dose  so  as  to  avoid  the  phenomenon  termed  "  deflec- 
tion of  complement."  Furthermore,  we  now  know 
that  the  defence  of  the  animal  body  against  bacterial 
invasion  is  not  solely  a  matter  of  bactericidal  and 
antitoxic  substances.  The  brilliant  studies  of  Ehr- 
lich,  Bordet,  and  others  on  the  humoral  side  of 
immunity  has  until  recently  caused  the  cellular 
side  advocated  by  Metchnikoff  to  be  much  neglected. 
Perhaps  the  recent  work  begun  by  A.  E.  Wright  on 
opsonins  may  lead  us  in  the  right  direction.  The 
therapeutic  results  thus  far  achieved  by  the  use  of 
bactericidal  immune  sera  certainly  show  that  much 
remains  to  be  done  in  the  study  of  immunity. 


PRECIPITINS 

Definition.  —  All  of  the  foregoing  experiments 
have  concerned  themselves  with  the  results  obtained 
by  injection  of  cellular  material  of  one  animal  into 
another.  In  the  further  study  of  this  subject, 
experiments  were  made  to  discover  what  happens 
when  dissolved  albuminous  bodies  of  one  species  are 
injected  into  animals  of  another  species.  This  line 
of  investigation  was  first  pursued  by  Tchistowitsch,1 
who  injected  rabbits  with  the  serum  of  horses  and 
of  eels.  On  withdrawing  serum  from  such  rabbits 
and  mixing  it  with  horse  or  eel  serum,  the  mix- 
ture became  cloudy,  owing  to  the  precipitation  of 
part  of  the  albumin  of  the  horse  or  eel  serum  by 
that  of  the  rabbit.  Normal  rabbit  serum  does  not 
possess  this  property.  Bordet  was  able  to  demon- 
strate that  the  same  thing  takes  place  if  rabbits  are 
treated  with  chicken  blood.  On  mixing  such  a 
serum  with  chicken  serum,  a  precipitate  formed. 
The  substances  which  develop  in  the  serum  by 
treating  an  animal  with  albuminous  bodies  of 
another  animal,  and  which  precipitate  these  albumins 
when  the  sera  of  the  two  animals  are  mixed,  are 

1  Tchistowitsch,  Annal.  Pasteur.  Vol.  xiii,  1899. 
106 


PRECIPITINS  lO/ 

called  precipitins.1  This  power  of  the  organism  to 
react  to  the  injection  of  foreign  dissolved  albuminous 
substances  has  been  found  to  be  very  extensive. 

Bacterial  Precipitins.  —  In  1897,  R.  Kraus  showed 
that  the  serum  of  a  rabbit  immunized  against 
typhoid  often  produces  a  precipitate  in  the  bac- 
terial-free filtrate  of  a  bouillon  culture  of  typhoid 
bacilli.  This  fact  has  been  verified  by  subsequent 
investigators  and  the  reaction  found  to  be  specific. 
In  general,  the  best  results  are  obtained  with  old 
bouillon  cultures  which  contain  a  larger  proportion 
of  the  autolytic  products.  It  was  natural  that  this 
reaction  should  at  once  be  applied  to  the  diagnosis 
of  typhoid  and  other  diseases.  Numerous  experi- 
ments however  have  shown  that  Kraus'  phenomenon 
is  not  nearly  so  constantly  observed  as  that  of 
agglutination,  and  the  reaction  is  therefore  but 
little  used.  Whether  the  bacterial  precipitins  are 
identical  in  character  with  those  obtained  by 
injecting  an  animal  with  an  unrelated  serum  (zoopre- 
cipitins),  is  still  undecided.  Rostoski,  as  well  as 
Nuttall,  believes  that  they  are  probably  different. 
So  much  for  bacterial  precipitins. 

Lactoserum  —  Other  Specific  Precipitins.  —  Bordet, 
by  injecting  cows'  milk  into  rabbits',  was  able  to 
produce  a  serum  which  precipitates  the  casein  of 

1  It  will  be  recalled  that,  besides  the  production  of  pre- 
cipitins, the  above  procedure  causes  the  formation  of  other 
anti-bodies  such  as  anti-complements,  anti-agglutinins,  etc. 


108  IMMUNE  SERA 

cows'  milk.  He  called  this  lactoserum.  Ehrlich, 
Morgenroth,  Wassermann,  Schutze,  Myers,  and 
Uhlenhuth  showed  that  by  treating  a  rabbit  with 
chicken  albumin  a  precipitin  is  formed  which  pre- 
cipitates chicken  albumin.  Myers,  by  treating  ani- 
mals with  Witte's  pepton  and  globulin,  produced  a 
serum  that  contained  specific  antipeptons  and  anti- 
globulins.  Pick  and  Spiro,  by  using  albumose, 
produced  antialbumoses.  Leclainche  and  Vallee, 
Stern,  Mertens,  and  Ziilzer  treated  animals  with 
human  albuminous  urine  and  produced  a  serum 
which  contained  a  precipitin  specific  for  this  sub- 
stance. Schutze,  by  treating  rabbits  with  a  vege- 
table albumin,  as  well  as  with  human  myoalbumin, 
produced  a  precipitin  specific  for  these  albumins. 
This  does  not  exhaust  the  recital  of  the  work  done 
in  this  field,  and  there  is  a  host  of  other  albuminous 
bodies  which,  when  injected  into  an  animal,  are 
able  to  excite  the  production  of  precipitins. 

Specificity  of  the  Precipitins.  —  It  was  soon  recog- 
nized that  the  specificity  is  not  absolute.  Above  all, 
this  depends  upon  the  strength  of  the  serum,  i.e., 
its  degree  of  activity.  This  is  measured  by  the 
dilution  in  which  it  will  still  react.  Thus  a  highly 
active  serum,  one,  for  example,  which  will  still 
give  a  distinct  reaction  when  diluted  i :  1000  or  over, 
will  produce  a  marked  precipitate  with  the  serum 
used  to  excite  its  production ;  whereas,  in  the  serum 
of  other  animal  species  it  will  produce  slighter  pre- 


PRECIP1TINS  109 

cipitates,  or  only  cloudings.  A  less  highly  active 
serum  will  likewise  cause  a  marked  precipitate  in 
the  homologous  blood  solution,  and  a  slight  pre- 
cipitate, or  only  a  clouding,  at  the  most,  in  a  closely 
related  species.  For  example,  the  serum  of  a  rabbit 
which  has  been  treated  with  sheep  blood  produces 
a  marked  precipitate  in  a  solution  of  sheep  blood ;  a 
slight  precipitate  in  a  goat -blood  solution ;  and  a  still 
fainter  one  in  an  ox-blood  solution.  In  some  in- 
stances the  two  latter  will  show  only  a  clouding.  If 
we  employ  a  very  weak  serum,  even  the  cloudings 
will  be  absent,  and  a  precipitate  is  formed  only  in 
the  sheep-blood  solution.  If  human  blood  or 
blood  serum  has  been  injected,  the  clouding  and 
precipitation  will  occur  most  readily  (aside,  of 
course,  from  human-blood  solution)  in  that  of  apes. 
In  the  precipitin  reaction,  therefore,  the  relationship 
of  the  single  animal  species  is  an  important  factor. 
This  peculiar  behavior  has  first  been  thoroughly 
studied  by  Nuttall l  who  made  observations  on 
five  hundred  different  animals.  As  a  result  of  these 
we  know  that  a  weak  human-blood  antiserum, 
besides  reacting  on  human  blood,  causes  a  clouding 
only  in  the  blood  of  anthropoid  apes  (chimpanzee, 
gorilla,  orang-outang) ;  a  stronger  serum  causes  a 
clouding  also  in  the  blood  of  other  monkeys ;  finally 

1  British  Medical  Journal,  1901,  Vol.  ii,  and  1902,  Vol.  i. 
See  also  Nuttall,  Blood  Immunity  and  Blood  Relationship, 
1904.  The  Macmillan  Co.,  N.  Y. 


1 10  IMMUNE  SERA 

a  very  highly  active  serum  reacts  with  the  blood  of 
all  the  mammalia.  In  that  case,  of  course,  only  a 
faint  clouding  is  produced  even  after  considerable 
time.  Nuttall  also  obtained  antisera,  each  of  which 
was  specific  for  one  of  the  large  animal  classes 
(birds,  reptiles,  amphibia).  Here,  too,  the  same 
quantitative  differences  were  noted. 

Nature  of  the  Precipitins.  —  The  precipitins  are 
fairly  resistant  bodies,  whose  power  gradually 
declines  at  a  temperature  of  60°  C.,  but  is  not  lost 
until  70°  C.  is  reached.  Once  their  action  is  lost, 
it  cannot  be  restored  by  the  addition  of  normal  sera, 
showing  that  the  precipitins,  like  the  agglutinins, 
are  receptors  of  the  second  order  and  are  not  ambo- 
ceptors.  The  resulting  precipitate  is  soluble  in 
weak  acids  and  alkalies.  Peptic  digestion  destroys 
the  substances  which  effect  the  precipitation. 
Leblanc  found  that  the  precipitins  were  precipitated 
from  the  serum  in  that  fraction  which  Hofmeister 
calls  the  pseudo  globulins.  Eisenberg,  on  the  other 
hand,  in  his  experiments  found  them  in  the  eu- 
globulin  fraction.  The  latter  result  was  also  obtained 
by  Obermayer  and  Pick  in  precipitins  obtained 
from  goats  and  rabbits.  The  discordant  results 
are  comprehensible  in  view  of  recent  publications 
concerning  the  unreliability  of  ammonium  sulphate 
fractionation  of  serum  globulins.  The  nature  of 
the  resulting  precipitate  has  also  been  studied  by 
Leblanc.  He  finds  that  it  is  a  combination  of  the 


PRECIPITINS  1 1 1 

precipitated  albumin  with  the  antibody  of  the 
specific  serum.  In  this  combination  the  properties 
of  the  pseudo  globulin  predominate  showing  that 
it  is  the  specific  serum  which  furnishes  the  greater 
part  of  the  precipitate.  The  presence  of  salts  seems 
to  be  necessary  for  the  precipitin  reaction.  A  tem- 
perature of  37°  C.  hastens,  while  a  low  temperature 
markedly  retards  the  reaction.  In  either  case,  the 
amount  of  precipitum  is  uninfluenced.  The  pres- 
ence of  even  small  quantities  of  acids  or  alkalies 
markedly  reduces  the  amount  of  precipitum  formed, 
but  an  increase  of  salt  (NaCl)  has  little  effect. 

Practical  Application.  -  -  These  precipitins  have 
very  recently  found  a  practical  application.  Fish, 
Ehrlich,  Morgenroth,  Wassermann,  and  Schutze 
investigated  the  specific  action  of  lactoserum.  They 
found  that  a  serum  derived  by  treating  an  animal 
with  cows'  milk  contained  a  precipitin  which  reacted 
only  on  the  casein  of  cows'  milk,  but  not  on  that  of 
human  milk  or  goats'  milk.  The  serum  of  an  ani- 
mal treated  with  human  milk  was  specific  for  the 
casein  of  human  milk,  etc.  Ehrlich,  Morgenroth, 
and  Wassermann  also  experimented  with  the  serum 
resulting  from  treatment  with  chicken  egg  albumin, 
and  found  that  this,  while  not  strictly  specific  so  far 
as  closely  related  species  are  concerned,  is  yet  so 
against  other  species.  The  precipitins,  therefore, 
react  on  closely  related  albumins,  but  are  specific 
against  those  of  unrelated  species. 


112  IMMUNE  SERA 

The  Wassermann-  Uhlenhuth  Blood  Test.  —  As  a 
result  of  these  researches  Wassermann  proposed, 
at  the  Congress  for  Internal  Medicine,  1900,  to  use 
these  sera  as  a  means  of  differentiating  albumins, 
i.e.,  to  distinguish  the  different  albumins  from  one 
another,  and  particularly  to  distinguish  those 
derived  from  man  from  those  of  other  animals. 
This  proposal  thus  to  use  the  Tchistowitsch-Bordet 
precipitins  had  important  practical  and  theoretical 
results.  Uhlenhuth,  Wassermann,  Schiitze,  Stern, 
Dieudonne,  and  others  showed  that  a  serum  could 
be  produced  from  rabbits  by  injecting  them  with 
human  serum,  by  means  of  which  it  is  possible  to 
tell  positively  whether  a  given  old,  dried  blood  stain 
is  human  blood  or  not. 

Uhlenhuth  1  tested  nineteen  kinds  of  blood  and 
only  obtained  a  reaction  with  human  blood  upon 
adding  antihuman  serum  to  the  series  of  dilutions. 
He,  moreover,  found  that  human  blood  which  had 
been  dried  four  weeks  on  a  board  could  be  readily 
distinguished  by  means  of  antihuman  serum  from 
the  blood  of  the  horse  and  ox.  On  the  following 
day  Wassermann  2  demonstrated  experiments  simi- 
lar to  Uhlenhuth 's  at  the  meeting  of  the  Physiologi- 
cal Society,  Berlin.  Outside  of  human  blood  only 
that  of  a  monkey  gave  the  reaction  with  anti- 
human  serum. 

1  Uhlenhuth,    Deutsche    med.    Wochenschrift,    1901.     xxvii. 

2  Wassermann  A.  and    Schutze,  Berliner  klin.  Wochenschr. 
1901.     No.  xxviii. 


PRECIPIT1NS  113 

The  reliability  of  this  reaction  in  medico-legal 
questions  has  been  abundantly  established.  In  the 
forensic  blood  diagnosis  the  subjects  of  the  test 
are  usually  blood  stains  on  clothing,  and  on  wood 
and  metal  objects.  After  such  a  doubtful  stain 
has  been  dissolved  in  physiological  salt  solution, 
one  first  proceeds  to  determine  that  it  is  really 
blood.  For  this  purpose  Teichmann's  test  (the 
production  of  ha3min  crystals),  the  guaiac  test,  and 
the  spectroscopic  examination  are  undertaken.  This 
is  of  considerable  importance,  for  not  merely  blood 
but  other  albuminous  solutions  derived  from  the 
same  animal  react  with  an  antiserum  obtained  by 
injecting  an  animal  with  blood  or  serum.  Having 
found  that  the  stain  is  that  of  blood,  we  next  deter- 
mine the  special  kind  of  blood. 

Immunizing  the  Animals.  —  For  the  production  of 
the  antisera,  we  make  use  of  rabbits.  These  can  be 
injected  either  with  sterile,  freshly-defibrinated  blood 
or  with  sterile  serum,  the  latter  being  preferable 
for  intravenous  inoculation.  It  is  well  to  begin 
with  small  doses  and  gradually  increase;  thus  for 
intravenous  inoculations  the  first  injection  should 
be  about  one  c.c.  and  increased  up  to  three  or 
four  c.c.  With  intraperitoneal  injections  about 
double  these  doses  can  be  given.  Ordinarily,  the 
interval  between  injections  is  three  or  four  days, 
and  the  entire  duration  of  treatment  from  two 
weeks  to  a  month.  Long-continued  treatment 


114  IMMUNE  SERA 

leads   to   a  disappearance   of   precipitins  from  the 
blood. 

Collecting  the  Serum.  —  When  the  animals  have 
received  five  to  six  injections,  and  some  days  have 
elapsed  it  is  well  to  draw  off  samples  of  the  blood 
and  to  test  for  precipitins.  This  is  easily  done  by 
shaving  the  ear  and  cleansing  the  skin  with  alcohol 
and  sterile  water.  An  incision  is  then  made  into 
the  marginal  vein  and  a  few  drops  of  blood  collected 
in  a  small  test-tube.  This  is  then  set  aside  to  allow 
the  blood  to  coagulate.  After  the  serum  has  sepa- 
rated it  can  be  tested  and  if  it  prove  insufficiently 
powerful,  treatment  may  be  continued,  otherwise 
the  animal  may  be  killed,  preferably  a  week  or  ten 
days  after  the  last  injection.  The  animals  may  be 
killed  in  a  variety  of  ways.  Uhlenhuth  chloroforms 
them,  opens  the  thoracic  cavity  under  aseptic 
precautions,  and,  cutting  through  the  beating  heart, 
the  blood  is  allowed  to  flow  into  the  thoracic  cavity, 
whence  it  is  removed  by  means  of  sterile  pipettes 
to  suitable  vessels.  Nuttall's  method  is  to  shave 
the  neck  and  disinfect  the  skin  with  lysol  solution; 
bend  the  animal's  head  backward  to  put  the  skin 
of  the  neck  on  the  stretch,  and  have  an  assistant 
make  a  clean  sweep  with  a  sterilized  knife  through 
the  tense  skin  to  and  through  the  vessels.  The 
blood  spurts  into  a  large  sterile  dish  which  is 
immediately  covered  when  the  main  flow  has  ceased. 
The  dishes  are  placed  horizontally  until  a  clot  has 


PRECIPITINS  1 1 5 

formed ;  they  are  then  slightly  tilted,  and  as  soon  as 
serum  enough  has  been  expressed,  this  is  pipetted 
off  into  sterile  test  containers  which  are  stored  in  a 
cool  place.  It  is  well  not  to  add  any  preservative 
to  the  serum,  as  such  an  addition  may  occasionally 
lead  to  pseudo  reactions. 

The  Test.  —  In  carrying  out  the  test  the  sus- 
pected clot  is  mixed  with  a  small  quantity  of  normal 
salt  solution  and  then  filtered.  Whether  or  not  the 
blood  specimen  has  gone  into  solution  can  best  be 
judged  by  the  foam  test.  Air  is  blown  gently  through 
the  pipette  which  is  used  for  transferring  the  solu- 
tion into  the  test-tubes.  Solutions  of  blood  or 
serum  of  i :  1000  and  over,  still  foam  well.  The  color 
of  the  fluid  is  not  so  reliable  an  index  of  solution. 
To  some  of  this  solution  in  a  test-tube,  about  double 
the  amount  of  the  specific  serum  (derived  as  above) 
is  added.  As  a  control  test,  we  place  a  little  blood 
of  another  species,  e.g.,  of  an  ox,  in  a  second  test- 
tube  together  with  some  of  the  specific  serum  and  a 
little  normal  salt  solution.  In  a  third  tube  we 
place  some  of  the  suspected  blood  solution,  and  in  a 
fourth  some  of  the  specific  serum  mixed  with  the 
normal  salt  solution.  All  four  tubes  are  placed  in 
the  incubator  at  37°C.  for  one  hour,  or  are  left  at 
room  temperature  for  several  hours.  If  the  sus- 
pected clot  was  one  of  human  blood,  the  first  tube 
will  show  distinct  evidence  of  precipitation,  while 
all  the  control  tubes  will  have  remained  clear.  It 


116  IMMUNE  SERA 

is  desirable  to  dilute  the  suspected  blood  as  far  as 
possible  when  testing,  for  when  concentrated  sera 
are  brought  together  reactions  may  occur  which 
will  lead  to  erroneous  conclusions.  In  medico- 
legal  work  it  will  be  well  to  progressively  dilute  a 
suspected  blood  sample  and  to  reach  a  conclusion 
upon  the  highest  (within  limits)  which  reacts  to  a 
given  antiserum.  In  routine  work  one  can  com- 
mence with  dilutions  of  the  suspected  blood  of 
i :  100  or  i :  200.  We  must  not  omit  to  say  that  it 
is  necessary  to  test  to  litmus  all  solutions  to  be 
examined,  and  to  neutralize  any  that  are  found 
decidedly  acid  or  alkaline. 

Appearance  of  the  Reaction. --When  antiserum 
is  added  to  blood  dilution  it  sinks  to  the  bottom  of 
the  tube,  forming  a  milky  white  zone  at  the  point  of 
contact.  The  milkiness  gradually  extends  upward 
until  the  whole  fluid  is  clouded.  Where  the  fluids 
have  been  mixed  by  shaking  this  diffuse  cloudiness 
undergoes  a  change;  after  ten  to  twenty  minutes, 
or  later,  very  fine  granules  of  precipitum  begin  to 
appear,  and  the  upper  layers  of  the  fluid  begin  to 
clear,  due  to  sedimentation  of  the  precipitum. 
The  fine  particles  soon  become  aggregated  into 
coarser  ones,  and  these  into  flocculi  which,  gradually 
sinking  to  the  bottom  of  the  tube,  give  rise  to  more 
or  less  deposit  of  a  whitish  appearance.  With  blood 
dilutions  of,  say  i :  40  to  i :  200  and  over,  the  deposit 
formed  is  usually  sharply  defined ;  where  more  con- 


PRECIPITINS  II/ 

centrated  dilutions  are  used,  the  deposit  may  form 
an  irregular  mass  at  the  bottom  of  the  tube. 

The  reaction  may  be  "followed  microscopically  by 
means  of  the  hanging-drop  method.  By  this  method 
a  reaction  can  be  observed  within  ten  to  fifteen 
minutes,  which  macroscopically  becomes  visible 
only  after  twt)  hours. 

Delicacy  of  the  Precipitin  Test.  -  -  Whereas  the 
ordinary  chemical '  tests  cease  to  give  reactions  in 
blood  dilutions  of  about  i :  1000,  powerful  antisera 
greatly  exceed  this  limit,  as  the  reported  results  of 
independent  observers  have  shown.  Working  with 
an  antihuman  serum,  Strube  reports  a  reaction 
with  a  blood  diluted  20,000  times,  and  Stern  one 
with  a  blood  diluted  50,000  times.  Ascoli  obtained 
a  reaction  with  a  specific  serum  with  egg  albumin 
diluted  1,000,000  times. 

Oilier  Applications  of  the  Precipitin  Test.  —  It  can 
be  readily  understood  that  this  test  finds  ready 
application  in  the  detection  of  horse,  dog,  or  cat 
meat  in  sausage. 

The  principle  and  the  method  are  the  same  in  all 
these  various  applications.  We  treat  animals  with 
the  albumins  which  we  wish  to  differentiate,  and  so 
obtain  sera  specific,  each  for  its  particular  kind  of 
albumin.  These  sera,  then,  produce  precipitates 
only  in  solutions  of  their  respective  albumins.  For 
example,  if  we  wish  to  determine  whether  a  given 
sample  of  meat  is  horse-flesh  or  not  we  must  inject 


Il8  IMMUNE  SERA 

an  animal  with  horse  serum,  or,  if  we  prefer,  with 
an  extract  of  horse-flesh.  The  serum  derived  from 
this  animal  will  then  produce  a  precipitate  in  the 
aqueous  extract  of  the  meat  if  this  be  horse-flesh, 
but  not  if  it  be  beef.  Animals  treated  with  dog 
serum  yield  a  serum  which  precipitates  an  aqueous 
extract  of  dog-flesh,  etc.  The  method  of  examina- 
tion consists  in  scraping  the  meat  and  extracting  it 
with  water  or  normal  salt  solution.  It  takes  a  long 
time  to  extract  the  meat  in  some  cases.  An  extract 
is  suitable  for  testing  when  it  foams  on  being  shaken. 
If  the  extract  is  very  cloudy  it  should  be  cleared  by 
filtration  through  a  Berkfeld  filter.  In  testing,  add 
ten  to  fifteen  drops  of  antiserum  to  3  cc.  of  the 
saline  meat  extract. 

Antiprecipitins  —  Iso-precipitins.  —  Biologically, 
the  precipitins  are  found  to  behave  like  the  sub- 
stances already  studied.  It  is  possible,  for  example, 
by  injecting  an  animal  with  a  precipitin,  say 
lactoserum,  to  obtain  an  antiprecipitin,  an  anti- 
lactoserum,  which  counteracts  or  inhibits  the  action 
of  the  precipitin.  This  is  entirely  analogous  to  the 
antihasmolysins,  the  antispermo toxin,  etc. 

If  rabbits  are  treated  with  rabbit  serum,  a  serum 
is  obtained  which  will,  in  certain  cases,  precipitate 
the  serum  of  other  rabbits.  This  was  done  by 
Schiitze,  and  he  called  this  serum  iso -precipitin. 


II.   CYTOTOXINS 

Cytotoxins  —  Definition  —  Leucotoxin  —  Nature  of 
the  Cytotoxin  —  Anticytotoxin.  -  -  After  it  had  been 
found  that  the  injection  of  an  animal  with  red  blood 
cells  of  another  animal  was  followed  by  the  produc- 
tion of  definite,  specific  reaction  substances,  investi- 
gators experimented  to  see  whether  this  was  also 
the  case  if  other  animal  cells  were  used.  Injections 
were  made  with  white  blood  cells,  spermatozoa  of 
other  animals,  etc.,  and  there  resulted  a  series  of 
reaction  substances,  entirely  analogous  to  the 
haemolysins,  which  were  specific  for  the  cells  used  for 
injection.  These  sera  Metchnikoff  calls  cytotoxins. 
After  Delezenne  had  published  a  short  article  on  a 
serum  haemolytic  for  white  blood  cells,  Metchnikoff 
undertook  a  study  of  the  substances  produced  in 
sera  of  animals  treated  with  leucocytes  of  another 
species.  He  injected  guinea  pigs  with  the  mesen- 
teric  glands  and  bone  marrow  of  a  rabbit.  He 
also  injected  for  several  weeks  half  an  Aselli's  pan- 
creas at  a  time,  at  intervals  of  four  days.  If  he 
withdrew  serum  from  such  a  guinea  pig  he  found 
this  to  be  intensely  solvent  for  white  blood  cells  of 
a  rabbit.  He  called  this  serum  leucotoxin.  This 
leucotoxin  is  very  poisonous  for  these  animals,  and 


120  IMMUNE   SERA 

kills  them  within  a  few  hours.  Non-fatal  doses  at 
first  excite  a  marked  hypoleucocytosis,  which  is 
followed  after  a  few  days  by  a  compensatory  hyper- 
leucocytosis.  Leucotoxin  destroys  the  mononu- 
clear  as  well  as  the  polynuclear  leucocytes  of  the 
animal,  as  was  shown  by  Funk.  Leucotoxin  which 
had  been  derived  by  injection  of  the  leucocytes  of 
horses,  oxen,  sheep,  goats,  or  dogs  acted  only  on 
the  leucocytes  of  that  species,  not  on  the  leucocytes 
of  man.  So  far  as  the  mechanism  of  the  cytotoxic 
action  is  concerned,  it  has  been  found  that  this  is 
the  same  as  that  of  the  haemolysins.  The  action  of 
the  specific  cytotoxic  serum  is  always  due  to  the 
combined  action  of  two  substances  in  the  serum,  a 
specific  immune  body,  and  an  alexin  or  comple- 
ment present  also  in  normal  serum.  The  cyto- 
toxic sera,  like  the  hsemolytic  sera,  are  rendered 
inactive  by  heating  to  55°  C.  In  other  respects 
also  the  cytotoxic  sera  maintain  the  analogy  to 
the  hasmolytic  sera.  Thus  it  is  possible  by  immu- 
nizing with  a  cytotoxin  to  obtain  an  anticytotoxin. 
Metchnikoff,  for  example,  was  able  to  produce  an 
antileucotoxin  by  injecting  animals  with  leuco- 
toxin.  This  antibody  inhibited  the  action  of  the 
leucotoxin. 

Neurotoxin.  —  Delezenne  and  Madame  Metchni- 
koff have  injected  animals  with  central-nervous- 
system  substance,  and  so  produced  a  specific  neuro- 
toxin.  They  injected  ducks  intraperitoneally,  giving 


CYTOTOXINS  121 

them  five  or  six  injections  of  ten  to  twenty  grammes 
of  dog  brain  and  spinal  cord  mixed  with  normal 
salt  solution.  The  serum  of  these  ducks  injected 
intracerebrally  into  dogs  in  doses  of  0.5  c.c. 
caused  the  dogs  to  die  almost  at  once  in  complete 
paralysis,  whereas  if  normal  duck  serum  was  in- 
jected in  the  same  way  no  effects  of  any  kind  were 
produced.  If  smaller  doses  of  the  specific  neuro- 
toxic  serum  were  administered,  say  o.i  to  0.2  c.c., 
various  paralyses  and  epileptiform  convulsions  set 
in,  from  which  the  animals  sometimes  recovered. 
The  action  of  this  serum  is  specific,  i.e.,  the  serum 
of  ducks  treated  with  dog  brain  causes  these  symp- 
toms only  in  dogs,  while  on  rabbits  it  acts  no 
differently  than  normal  duck  serum. 

Spermatoxin.  —  Another  specific  cell-dissolving 
serum  was  produced  by  Landsteiner,  Metchnikoff, 
and  Moxter,  by  injecting  animals  with  the  sperma- 
tozoa of  other  animals.  Such  a  serum  rapidly 
destroys  the  spermatozoa  of  the  animals  whose 
product  was  injected.  This  cytotoxin  was  named 
spermatoxin.  If  animals  are  treated  with  spermato- 
zoa there  is  produced  a  serum  which  is  not  only  a 
spermatoxin,  but  which  is  also  hasmolytic  for  the 
red  cells  of  that  animal.  This  was  demonstrated 
by  Metchnikoff  and  Moxter,  and  has  already  been 
referred  to  in  discussing  haemolysins.  If,  for  ex- 
ample, we  inject  the  spermatozoa  of  sheep  into 
rabbits,  we  shall  obtain  a  serum  that  is  sperma- 


122  IMMUNE  SERA 

toxic  for  sheep,  as  well  as  hasmolytic  for  sheep 
red  cells. 

Common  Receptors.  —  At  first  it  was  thought  that 
the  hasmolysin  so  produced  was  due  to  the  presence 
of  small  quantities  of  blood  injected  with  the  sper- 
matozoa. The  same  result  however  was  obtained 
when  all  traces  of  blood  could  be  excluded  j1  further- 
more a  number  of  investigators  produced  hsemoly- 
sins  by  the  injection  of  fluids  entirely  free  from  red 
corpuscles,  such  as  serum  and  urine.  The  produc- 
tion of  this  haemolysin  is  not  hard  to  explain  if  we 
hold  fast  to  the  side-chain  theory.  We  have 
merely  to  assume  that  the  spermatozoa  or  these 
other  substances  possess  certain  receptors  in  com- 
mon with  the  red  blood  cells  of  the  same  animal. 
Ehrlich  and  Morgenroth  2  have  repeatedly  pointed 
out  that  specificity  is  a  matter  not  of  cells,  but  of 
receptors.  Despite  these  very  conclusive  demon- 
strations later  investigators,  who  attempted  to 
produce  antisera  for  the  cells  of  various  organs, 
continued  to  use  emulsions  of  unwashed  organs,  in 
utter  disregard  of  the  presence  of  free  receptors  in 
the  organ  juices  and  also  without  consideration  of 
the  antibodies  certain  to  be  produced  by  the  red 
cells  normally  present. 

Cytotoxin  for  Epithelium.  —  As  far  back  as   1899, 

1  Von  Dungern.     See  Collected  Studies  on  Immunity,  p.  47. 
Wiley  and  Sons,  New  York,  1906. 

2  Ehrlich  and  Morgenroth,     Ibid.,  p.  IPO. 


CYTOTOXINS  123 

von  Dungern  showed  that  it  was  possible  to  produce 
an  antiepithelial  serum  by  treating  animals  with 
the  ciliated  tracheal  epithelium  of  oxen.  This 
serum  was  rapidly  destructive  for  this  particular 
kind  of  epithelium,  but  it  contained  also  a  specific 
haemolytic  body  just  as  was  the  case  in  the  sper- 
motoxic  serum,  and  for  the  same  reasons.  This 
antiepithelial  serum  aroused  considerable  interest 
since  it  indicated  the  possibility  of  producing  sera 
which  were  cytotoxic  for  certain  varieties  of  epi- 
thelial cells,  especially  those  of  pathological  origin, 
as  carcinoma.  The  numerous  experiments  made 
in  this  direction  failed  however  to  produce  the 
desired  results.  Owing  to  the  extensive  distribu- 
tion of  common  receptors  the  antisera  were  found 
to  exhibit  quite  general  properties  and  to  lack 
that  degree  of  cell  specificity,  essential  for  practical 
purposes. 

Cytotoxins  by  the  Use  of  Nucleo-Proteids.  --In 
order  to  prevent  the  adventitious  formation  of 
those  bodies  resulting  from  impure  methods  of 
immunization,  and  also  in  the  hope  of  obtaining 
greater  specificity,  a  few  investigators  have  utilized 
the  nucleo-proteids  of  the  cell  for  immunization. 
This  method  seems  to  have  been  tried  first  by 
Marrassini  in  1903,  but  with  indifferent  results. 
In  1905  Beebe  *  published  an  extensive  study  along 

1     S.   P.   Beebe,  Cytotoxic  Serum  Produced  by  the  Injection 
of  Nucleo-Proteids,     Journ.  Exper?  Medicine,  Vol  vii,  1905. 


124  IMMUNE   SERA 

these  lines  and  described  the  formation  of  a  nephro- 
toxic  serum  which  caused  albuminuria  and  acute 
degeneration  of  the  kidney  without  changes  in 
the  other  organs.  Albuminuria  appeared  gene- 
rally on  the  fourth  or  fifth  day,  increased  rapidly 
in  amount,  and  was  accompanied  by  the  excretion 
of  hyaline  and  granular  casts.  Recently  Pearce 
and  Jackson,1  after  a  careful  experimental  study  on 
the  production  of  cytotoxic  sera  by  the  injection  of 
nucleo-proteids,  conclude  "  that  the  results  do  not 
support  the  theory  that  specific  cytotoxic  sera  may 
be  developed  in  this  way,  but  indicate,  rather,  that 
such  sera  have  certain  mildly  toxic  properties  acting 
in  a  general  way  and  affecting  especially  the  principal 
excretory  organ,  the  kidney." 

1  R.  M.  Pearce  and  Holmes  Jackson,  Journal  of    Infectious 
Diseases,  Vol.  iii,   1906. 


OPSONINS  OR  BACTERIOTROPIC 
SUBSTANCES 

Historical.  -  -  The  early  work  of  Nuttall  and 
others  on  the  bactericidal  action  of  normal  serum, 
and  Pfeiffer's  demonstration  of  the  bacteriolysis 
of  cholera  and  typhoid  bacilli  by  immune  sera  in  the 
absence  of  cells,  formed  the  chief  basis  on  which 
rested  the  humoral  theory,  which  attributed  the 
protection  in  such  cases  to  the  destructive  action 
of  the  serum  on  the  microbes.  It  was  found,  how- 
ever, that  cases  of  protection  resulting  from  the 
use  of  immune  serum  occurred  where  no  such 
bacteriolytic  action  could  be  demonstrated;  infec- 
tion with  plague  or  streptococcus  may  be  men- 
tioned as  examples.  It  is  now  pretty  generally 
accepted  that  immunity  in  these  cases  is  due  largely 
to  the  phagocytic  action  of  the  leucocytes.  As  far 
back  as  1858  Haeckel  had  observed  that  particles 
of  indigo  injected  into  the  veins  of  certain  molluscs 
could  shortly  afterwards  be  found  in  the  blood 
cells  of  the  animal.  However,  the  significance  of 
this  and  other  observations  was  not  appreciated 
until  Metchnikoff 1  in  1883  called  attention  to  their 
bearing  on  infection  and  immunity.  The  outcome 

1  Arbeiten  des  Zoo  log.  Institutes  in  Wien,   1883,  Vol.  v. 

125 


126  IMMUNE  SERA 

of  his  investigations  was  the  establishment  of  the 
well-known  doctrine  of  phagocytosis,  the  principle 
of  which  is  that  the  wandering  cells  of  the  animal 
organism,  the  leucocytes,  possess  the  property  of 
taking  up,  rendering  inert,  and  digesting  micro- 
organisms which  they  may  encounter  in  the  tissues. 
MetchnikofT  believes  that  susceptibility  to  or 
immunity  from  infection  is  essentially  a  matter 
between  the  invading  bacteria  on  the  one  hand 
and  the  leucocytes  of  the  tissues  on  the  other.  He 
realizes  that  the  serum  constituents  play  an  im- 
portant role,  but  this  role  consists  in  their  stimulat- 
ing the  leucocyte  to  take  up  the  bacteria. 

Thus  if  a  highly  virulent  organism  is  injected 
into  a  susceptible  animal,  the  leucocytes  appear  to 
be  repelled,  and  to  be  unable  to  deal  with  the 
microbe,  which  multiplies  and  causes  the  death  of 
the  animal.  If,  however,  the  suitable  immune 
serum  is  injected  into  the  animal  before  inoculation, 
the  phagocytes  attack  and  devour  the  invading 
micro-organisms.  Admitting  that  the  phagocyte 
plays  an  important  part  in  certain  infections  the 
question  must  still  be  considered  whether  the 
immune  serum  has  acted  on  the  injected  microbes 
or  on  the  phagocytes.  Metchnikoff,  we  have  seen, 
takes  the  latter  view. 

In  1903  A.  E.  Wright  *  called  attention  to  certain 
substances  present  in  serum  which  acted  on  bacteria 

1  Wright  and  Douglas,  Proc.  Royal  Society,  Vol.  72,  1903. 


OPSONINS  127 

and  rendered  them  more  easily  taken  up  by  the 
phagocytic  cells.  He  called  this  substance  opsonin 
and  showed  that  it  is  present  in  normal  as  well  as 
immune  sera.  By  means  of  absorption  tests 
modelled  after  those  of  Ehrlich  and  Morgenroth,  he 
showed  that  the  opsonin  has  a  specific  affinity  for 
the  bacteria  and  none  for  the  leucocytes.  The 
opsonins  for  staphylococcus  prepare  only  staphy- 
lococci  for  the  leucocytes,  those  for  tubercle  bacilli 
only  these  bacteria,  etc.  As  a  result  of  his  obser- 
vations Wright  supposes  that  the  phagocytes  play 
only  a  passive  role,  which  depends  on  the  pre- 
liminary action  of  the  opsonin. 

Bacteriotropic  Substances.  -  -  Independently  of 
Wright,  though  somewhat  later,  Neufeld  and  Rim- 
pau  *  of  Berlin  published  experiments  on  the  pha- 
gocytic effect  of  immune  sera.  They  also  found 
that  in  these  sera  there  exists  a  substance  which  has 
no  direct  action  on  the  phagocytes,  but  which  can 
fix  itself  on  the  corresponding  bacteria  and  so  modify 
these  that  they  are  more  readily  devoured  by  the 
phagocytes.  They  call  this  constituent  a  "  bacte- 
riotropic  substance."  There  is  little  doubt  that  this 
bacteriotropic  substance  and  Wright's  opsonin  are 
identical.  Certain  differences  in  the  effect  of  heat 
are  probably  to  be  explained  by  the  differences  in 
the  quantities  of  these  sensitizing  substances  in 
normal  and  immune  sera. 

1  Neufeld  and  Rimpau,  Deutsche  med.  Wochenschrift,  1904. 


>£&*!*-•        ^^X 

f  GF   THE  X 

HVERSITY  ) 
// 


128  IMMUNE  SERA 

Opsonins  Distinct  Antibodies.  —  It  was  natural  to 
question  whether  these  "  opsonins  "  were  really  dis- 
tinct from  other  antibodies,  or  whether  they  were 
perhaps  identical  with  the  immune  body  (or  sub- 
stance sensibilatrice).  In  a  series  of  papers  on  this 
subject  Hektoen  *  shows  that  the  former  is  the  case, 
opsonins  are  distinct  substances.  This  is  not  only 
indicated  by  the  results  of  absorption  tests,  but  by 
the  fact  that,  by  immunization,  a  serum  can  in  cer- 
tain cases  be  obtained  which  is  opsonic  but  not  lytic, 
or  in  other  cases  one  which  is  lytic  but  not  opsonic. 
Similar  experiments  have  differentiated  opsonins 
from  agglutinins. 

Structure  of  Opsonins.  —  Opsonins,  like  agglu- 
tinins and  precipitins,  appear  to  possess  two  groups, 
opsoniferous  and  haptophore.  On  heating  an  op- 
sonic  serum  the  former  group  is  destroyed  but  the 
haptophore  group  remains  intact,  as  can  be  seen  from 
suitable  combining  experiments.  There  is  still  con- 
siderable difference  of  opinion  as  to  the  degree  of 
heat  necessary  to  inactivate  the  opsonins.  Once  the 
opsoniferous  group  has  been  destroyed  it  is  impos- 
sible to  restore  the  opsonic  action  by  the  addition 
of  a  complementing  substance.  Hence  the  opsonins 
are  to  be  regarded  as  receptors  of  the  second  order 
and  similar  in  structure  to  the  agglutinins  and 
precipitins. 

The  Opsonic  Index.  —  In  the  study  of  these  opso- 

1  Hektoen,  L.,  Journal  Infect.  Diseases,  1905  and  1906. 


OPSONINS  129 

nins  Wright  developed  the  idea  that  they  were 
highly  important  in  combating  a  number  of  bacterial 
infections,  such  as  staphylococcus  and  tubercle. 
His  observations  showed  that  inoculations  of  the 
corresponding  bacteria  produced  marked  changes  in 
the  opsonic  contents  of  the  infected  individual  and 
that  it  was  possible  to  estimate  accurately  the  im- 
munizing effect  of  such  inoculations. 

Technique.  —  Wright's  technique  of  measuring  the 
opsonic  power  is  a  slight  modification  of  the  Leish- 
man l  method  and  is  as  follows :  An  emulsion  of 
fresh  human  leucocytes  is  made  by  dropping  twenty 
drops  of  blood  from  a  finger  prick  into  20  c.c. 
normal  salt  solution  containing  one  per  cent  sodium 
citrate.  The  mixture  is  centrifuged,  the  supernatant 
clear  fluid  removed  and  the  upper  layers  of  the  sedi- 
mented  blood  cells  transferred  by  means  of  a  fine 
pipette  to  10  c.c.  normal  salt  solution.  After  cen- 
trifuging  this  second  mixture  the  supernatant  fluid 
is  pipetted  off  and  the  remaining  suspension  used 
for  the  opsonic  tests.  Such  a  "  leucocyte  emulsion,  ' 
of  course,  contains  an  enormous  number  of  red 
blood  cells;  the  proportion  of  leucocytes,  however, 
is  greater  than  in  the  original  blood. 

One  volume  of  this  emulsion  is  mixed  with  one 
volume  of  the  bacterial  suspension  to  be  tested  and 
with  one  volume  of  the  serum.  This  is  best  accom- 
plished by  means  of  a  pipette  whose  end  has  been 

1   Leishman,  British  Medical  Journal,  Jan.,  1902. 


130  IMMUNE  SERA 

drawn  out  into  a  capillary  tube  several  inches  in 
length.  With  a  mark  made  about  three-quarters 
of  an  inch  from  the  end  it  is  easy  to  suck  up  one  such 
volume  of  each  of  the  fluids,  allowing  a  small  air 
bubble  to  intervene  between  each  volume.  All 
three  are  now  expelled  on  a  slide  and  thoroughly 
mixed  by  drawing  back  and  forth  into  the  pipette. 
Then  the  mixture  is  sucked  into  the  pipette,  the  end 
sealed  and  the  whole  put  into  the  incubator  at  37°  C. 
The  identical  test  is  made  using  a  normal  serum  in 
place  of  the  serum  to  be  tested.  Both  tubes  are 
allowed  to  incubate  fifteen  minutes  and  then  ex- 
amined by  means  of  smear  preparations  on  slides 
spread  and  stained  in  the  usual  way.  The  degree  of 
phagocytosis  is  then  determined  in  each  by  count- 
ing a  consecutive  series  of  fifty  leucocytes  and  find- 
ing the  average  number  of  bacteria  ingested  per 
leucocyte.  This  number  for  the  serum  to  be  tested 
is  divided  by  the  number  obtained  with  the  normal 
serum  and  the  result  regarded  as  the  opsonic  index 
of  the  serum  in  question.  The  presence  of  a  high 
opsonic  index  Wright  regards  as  indicative  of  in- 
creased resistance.  He  further  states  that  the  fluc- 
tuation of  the  opsonic  index  in  normal  healthy 
individuals  is  not  more  than  from  .8  to  1.2,  and  that 
an  index  below  .8  is  therefore  almost  diagnostic  of 
the  presence  of  an  infection  with  the  organism  tested. 
Application  of  the  Opsonic  Measurements.  —  At 
the  present  time  Wright  has  correlated  all  his  obser- 


OPSONINS  131 

vations  and  built  up  a  system  of  treating  bacterial 
infections  by  means  of  active  immunization  con- 
trolled by  opsonic  measurements.  The  principles 
underlying  his  method  may  be  briefly  summarized 
as  follows:  In  localized  bacterial  infections  the 
infected  body  absorbs  but  small  amounts  of  bacterial 
substances  or  antigens.  In  consequence  of  this  the 
amount  of  active  immunity  developed  is  but  slight. 
Localized  infections  therefore  tend  to  run  a  chronic 
course.  The  logical  method  of  effecting  a  cure  in 
these  cases  is  to  actively  immunize  the  body  with 
the  invading  organism.  In  a  number  of  infections, 
notably  those  of  staphylococcus,  streptococcus,  and 
tubercle,  the  degree  of  immunity  is  measured  accu- 
rately by  the  opsonic  index.  Following  an  inocu- 
lation with  the  infecting  bacteria  (dead  cultures  in 
salt  solution)  there  is  first  a  drop  in  the  opsonic 
index,  the  "  negative  phase,"  then,  depending  on 
the  size  of  the  dose  and  the  reacting  power  of  the 
individual,  there  comes  a  rise  of  the  index,  the 
"  positive  phase,"  or  a  continuation  of  the  negative 
phase.  The  fbrmer  is  obtained  with  proper  dosage ; 
the  latter  with  doses  too  large  or  too  small.  In 
estimating  the  size  of  the  dose  given,  Wright  counts 
the  number  of  bacteria  per  c.c.  of  emulsion  injected. 
Thus  in  the  case  of  localized  staphylococcus  infec- 
tions the  doses  for  adult  humans  range  from  100 
million  to  500  million  bacteria.  In  the  case  of  step- 
tococcus  the  doses  are  smaller,  averaging  about  50 


132  IMMUNE  SERA 

to  100  million.  The  bacterial  suspensions  are  heated 
to  60°  C.  for  twenty  minutes,  0.5%  carbolic  acid 
is  added,  and  tests  are  made  to  insure  sterility. 
The  time  for  inoculation  is  governed  by  the  opsonic 
index.  If  the  first  inoculation  has  been  properly 
gauged  there  is  a  brief  negative  phase,  followed  by 
a  positive  phase  of  some  days'  duration.  As  this 
positive  phase  gradually  drops,  one  gives  another 
inoculation  and  watches  the  effect  on  the  opsonic 
index.  If  the  index  drops  markedly  and  rises  but 
little,  the  dose  has  been  too  large.  Or  if  the  nega- 
tive phase  is  slight,  and  the  positive  phase  slight  and 
transitory,  the  dose  has  been  too  small.  With 
proper  dosage  the  negative  phases  are  small,  and  the 
opsonic  index  is  kept  fairly  well  above  normal. 
Hand  in  hand  with  this  goes  an  improvement  in  the 
clinical  symptoms. 

Wright  and  his  pupils  have  published  accounts  of 
a  large  number  of  cases  successfully  treated  accord- 
ing to  this  method.  The  results  are  reported  as  espe- 
cially good  in  cases  of  severe  acne,  multiple  boils, 
lupus,  tubercular  glands,  and  bone  tuberculosis. 

In  judging  of  the  value  of  Wright's  method  we 
must  bear  clearly  in  mind  that  the  essential  feature 
of  it  is  the  control  by  opsonic  measurements;  treat- 
ment of  bacterial  infections  by  the  inoculation  of 
dead  cultures  has  long  been  known. 

The  results  obtained  by  most  workers  in  this  coun- 
try fail  to  bear  out  Wright's  claims  for  the  method. 


OPSONINS  133 

Thus  the  author l  finds  that  the  variation  in  the 
opsonic  indices  of  several  normal  persons  is  often 
considerable;  that  opsonic  counts  based  on  fifty 
leucocytes  may  occasionally  vary  by  more  than 
50%  and  that  it  is  therefore  necessary  to  count  from 
150  to  200  leucocytes  for  each  test;  that  duplicate, 
triplicate  and  more  tests  made  of  the  same  serum, 
at  the  same  time,  and  under  identical  conditions  so 
far  as  one  can  tell,  frequently  give  widely  divergent 
results;  that  the  opsonic  index  and  the  clinical 
course  of  the  disease  do  not  always  run  parallel. 
Cases  may  do  very  well  and  have  the  index  remain 
low;  other  cases  may  do  poorly  with  an  increased 
opsonic  index.  It  is  to  be  noted,  furthermore,  that 
some  of  these  variations  in  results  are  unavoidable, 
at  least  with  the  present  technique. 

To  one  who  has  followed  the  progress  of  immunity 
studies,  it  is  not  at  all  surprising  to  find  that  the 
opsonic  index  is  not  necessarily  a  measure  of  the 
patient's  immunity.  When  Gruber  and'  Durham 
published  their  observations  on  agglutinins  the 
phenomenon  was  at  once  hailed  and  interpreted  by 
many  as  measuring  the  degree  of  immunity  possessed 
by  the  patient.  The  same  error  was  made  when 
some  time  later  the  bacteriolytic  substances  were 
discovered.  In  both  cases  it  was  soon  found  that 
these  were  but  accompaniments  of  greater  or  less 
significance  to  the  complex  phenomenon  of  immun- 
1  Bolcluan,  Long  Island  Med.  Journal,  VoJ.  i,  1907, 


134  IMMUNE  SERA 

ity.  When  we  consider  how  manifold  are  the  defen- 
sive agencies  which  the  animal  organism  possesses, 
and  how  very  complex  they  become  the  more  they 
are  studied,  we  shall  not  marvel  at  the  absence  of 
parallelism  between  the  clinical  course  of  the  disease 
and  the  opsonic  index.  There  is  little  doubt  that 
the  opsonic  indices  do  measure  a  certain  fraction  or 
phase  of  the  immunity  reaction;  we  do  not  believe 
that  they  replace  clinical  observations  in  measuring 
the  effect  of  immunizing  injections. 


VII.  SNAKE  VENOMS  AND  THEIR  ANTISERA 

Despite  the  fact  that  venomous  serpents  have 
excited  the  fear  and  interest  of  mankind  for  centuries 
it  is  only  very  recently  that  we  have  come  to  know 
anything  definite  about  their  poisons.  This  is 
perhaps  in  part  due  to  the  fact  that  Europe  possesses 
but  few  poisonous  snakes,  and  so  offered  little 
material  for  study.  Some  idea  of  the  importance 
of  the  subject  for  certain  countries,  however,  can  be 
seen  when  it  is  stated  that  in  India  more  than 
20,000  persons  annually  die  from  the  bite  of  the 
hooded  cobra.  It  was  quite  natural,  therefore,  that 
one  of  the  earliest  modern  researches  into  the 
nature  of  snake  venom,  that  of  Calmette,1  should 
have  come  from  that  country.  This  author  also 
found  that  he  could  produce  an  antitoxic  serum  by 
injecting  animals  with  the  snake  venom. 

The  Venoms.  —  Our  present  knowledge  of  snake 
venoms  and  their  antisera  is  due  largely  to  the 
researches  of  Flexner  and  Noguchi  2  and  of  Kyes 
and  Sachs.3  The  venoms  of  different  snakes  vary 

1  Calmette,    Annal.    Inst.    Pasteur,    Vol.    vi,    1892;    Comptes 
rend.  Soc.  Biol.,   1894. 

2  Flexner  and  Noguchi,  Journal  Exp.  Medicine,  1902,  et  seq. 

3  Kyes  and  Sachs.   See  in  Collected  Studies   on   Immunity, 
Ehrlich,  New  York,  1906. 

135 


136  IMMUNE   SERA 

a  great  deal  in  their  toxic  properties,  and  this  is 
due  to  their  relative  contents  of  different  consti- 
tuents, as  follows:  —  haemagglutinins,  hsemolysin, 
haemorrhagin,  and  neurotoxin.  The  first  two  act 
exclusively  on  the  blood  cells,  the  hsemorrhagin  on 
the  endothelium  of  the  blood  vessels,  and  the 
neurotoxin  on  the  cells  of  the  central  nervous 
system.  The  last  named  causes  death  by  paralysis 
of  the  cardiac  and  respiratory  centers.  The  ven- 
oms of  the  cobra,  water-moccasin,  daboia  and 
some  poisonous  sea  snakes  are  essentially  neuro- 
toxic,  although  they  have  strong  dissolving  powers 
for  the  erythrocytes  of  some  animals.  In  study- 
ing the  haemolytic  powers  of  the  venoms  of  cobra, 
copperhead,  and  rattlesnake,  Flexner  and  Noguchi 
found  cobra  venom  to  be  the  most  haemolytic  and 
that  of  rattlesnake  the  least.  They  attribute  the 
toxicity  of  rattlesnake  poison  chiefly  to  the  action 
of  haemorrhagin.  The  venoms  of  the  water  mocca- 
sin and  the  copperhead  also  contain  hgemorrhagin. 

Unlike  the  bacterial  toxins  the  action  of  the  snake 
venoms  is  preceded  by  no  appreciable  incubation 
period.  In  addition  to  this  the  poisons  are  very 
rapidly  absorbed.  Thus  Calmette  found  that  a  rat 
inoculated  into  the  tip  of  the  tail  could  not  be  saved 
by  amputating  the  tail  one  minute  later.  Such 
animals  died  within  about  five  minutes  of  the  time 
required  for  control  animals. 

The  hasmolysin  and  neurotoxin  and  perhaps  also 


SNAKE   VENOMS  AND   THEIR  ANTISERA      137 

the  other  cytotoxic  substances  of  venom  consist  of 
amboceptors  which  find  a  complement  in  the  body 
of  the  poisoned  animal.  Not  only  does  ordinary 
serum-complement  serve  for  activation,  but,  accord- 
ing to  Noguchi,1  the  fatty  acids  contained  in  the  red 
blood  cells  also  act  as  complement. 

Antivenins.  —  Calmette  was  the  first  to  produce 
an  antiserum  against  snake  venom,  utilizing  for 
this  purpose  rabbits.  He  began  with  injections  of 
sV  of  a  fatal  dose,  and  injected  gradually  increasing 
doses  until  at  the  end  of  four  or  five  weeks  the 
animals  tolerated  double  a  fatal  dose.  By  con- 
tinuing the  treatment  he  finally  got  the  animals 
to  stand  80  fatal  doses  (40  mg.)  without  any 
reaction  whatever.  Five  drops  of  the  serum  of  such 
an  animal  neutralized  i  mg.  cobra  poison.  It 
has  been  found  that  anticobra  serum  protects 
against  the  neurotoxic  components  of  other  snake 
venoms,  furthermore  against  scorpion  poison  and 
the  poison  of  eel  blood.  The  serum  also  contains 
an  antihaemolysin,  but  no  antibody  against  haemor- 
rhagin  (of  the  rattlesnake).  It  is  therefore  without 
effect  on  rattlesnake  venom.  Antivenin  for  the 
latter  may  be  prepared  by  immunizing  goats  with 
corresponding  venoms  which  have  been  attenuated 
by  weak  acids.  Such  a  serum,  of  course,  possesses 
no  antineurotoxin  and  is  therefore  useless  against 
cobra  and  viper  venoms. 

*  Noguchi,  Jpurn.  Exper.  Medicine,  Vol,  ix,  1907. 


VIII.    SERUM   SICKNESS 

Definition.  —  Under  this  name  we  now  include 
the  various  clinical  manifestations  following  the 
injection  of  horse  serum  into  man.  The  princi- 
pal symptoms  of  this  disease  are  a  period  of  incu- 
bation varying  from  eight  to  thirteen  days,  fever, 
skin  eruptions,  swelling  of  the  lymph  glands, 
leukonemia,  joint  symptoms,  oedema  and  albumin- 
uria.  The  term  "  serum  sickness  "  was  first  used 
by  von  Pirquet  and  Schick,1  from  whose  excellent 
monograph  the  following  data  are  chiefly  taken. 

In  1874  Dallera  reported  that  urticarial  eruptions 
may  follow  the  transfusion  of  blood.  Neudorfer 
and  also  Landois  also  refer  to  this  complication. 
In  the  year  1894  the  use  of  diphtheria  antitoxin 
introduced  the  widespread  practice  of  injecting 
horse  serum.  In  the  same  year  several  cases  were 
reported  in  which  these  injections  were  followed  by 
various  skin  manifestations,  mostly  of  an  urticarial 
character.  Following  these  came  a  great  mass  of 
evidence  which  made  it  clear  that  following  the  in- 
jection of  antidiphtheric  serum  these  sequelae  were 
usually  comparatively  harmless.  Nevertheless  from 
time  to  time  the  occurrence  of  serious  symptoms, 

1  v.  Pirquet  and  Schick,  Die  Serum  Krankheit,  Wien,  1905. 

.38 


SERUM  SICKNESS  139 

and  even  of  death,  have  been  reported  following 
the  injection  of  diphtheria  antitoxic  serum.  Rose- 
nau  and  Anderson  have  collected  nineteen  such 
sudden  death  cases  from  the  literature,  and  state 
they  have  personal  knowledge  of  several  more 
which  have  not  been  reported.  However,  con- 
sidering the  enormous  number  of  antitoxic  injec- 
tions made  each  year,  such  accidents  must  be 
extremely  rare.  Certainly  the  benefits  derived  from 
diphtheria  antitoxin  far  outweigh  the  danger. 
In  over  50,000  persons  injected  in  New  York,  but 
two  deaths  attributed  to  the  serum  furnished  by 
the  Health  Department,  have  occurred. 

Due  to  Serum  as  Such.  —  Heubner  in  1894  and 
von  Bokay  somewhat  later  expressed  the  opin- 
ion that  these  manifestations  were  due  to  other 
properties  than  the  antitoxin  in  the  serum,  and 
this  has  proven  to  be  the  case.  Johannessen  pro- 
duced the  same  effects  by  injecting  normal  horse 
serum.  It  has  also  been  shown  that  the  skin  erup- 
tions and  other  symptoms  follow  in  direct  propor- 
tion to  the  amount  of  serum  injected,  and  this  has 
led  to  attempts  to  concentrate  the  serum  as  much 
as  possible.1  Park  has  also  shown  that  the  individ- 
uality of  the  horse  plays  an  important  r61e,  some 
horses  yielding  a  serum  which  gives  rise  to  a  large 
proportion  of  "  rashes." 

1  See  Gibson,  The  Concentration  of  Diphtheria  Antitoxin, 
Jour,  of  Biological  Chemistry,  Vol.  i,  1906. 


140  IMMUNE  SERA 

Von  Pirquet  and  Schick's  Theory.  —  It  was  diffi- 
cult to  account  for  the  long  incubation  period  in 
"  serum  sickness."  With  poisons  capable  of  self- 
multiplication  (bacteria,  etc. )  this  period  was  usually 
referred  to  the  time  necessary  for  them  to  accumu- 
late in  sufficient  number  and  virulence  to  produce 
symptoms.  But  serum  is  not  a  poison  capable  of 
multiplication.  Pfeiffer's  work  on  the  endotoxins 
led  to  the  view  that  the  antibodies  played  an  impor- 
tant part  in  bringing  on  the  symptoms  by  setting 
free  the  endotoxins.  The  results  of  these  observa- 
tions are  very  closely  related  to  von  Pirquet  and 
Schick's  explanation  of  the  production  of  serum 
disease.  The  endotoxic  theory,  in  the  sense  of  bac- 
teriolysis, naturally  cannot  be  applied  to  albumi- 
nous substances  in  solution.  We  can  only  accept 
it  in  the  sense  that  by  means  of  the  reaction  between 
the  antibodies  and  the  antigen  the  poisonous  sub- 
stance is  formed. 

It  is  of  course  at  once  apparent  that  the  formation 
of  antibodies  requires  a  definite  period  of  time.  The 
general  idea  underlying  von  Pirquet  and  Schick's 
theory  of  serum  sickness  is  that  the  injection  of  the 
horse  serum  into  man  causes  the  development  of 
specific  reaction  products  which  are  able  to  act 
upon  the  antigens  introduced.  These  antibodies 
encounter  the  antigens,  i.e.,  some  of  the  serum  still 
present  in  the  body,  and  so  give  rise  to  a  poisonous 
substance.  This  accounts  also  for  the  cases  of 


SERUM  SICKNESS  141 

"  immediate  reaction  "  described  by  von  Pirquet 
and  Schick,  in  which  second  injection  of  a  serum 
produces  an  attack  of  serum  sickness  without  any 
period  of  incubation.  This  includes  also  some  of 
the  cases  of  sudden  death  following  the  injection 
of  horse  serum.  Here  the  second  injection  comes 
at  a  time  when  the  accumulation  of  antibodies  is 
at  its  height.  Similar  results  were  obtained  in- 
dependently by  Rosenau  and  Anderson,1  who  found 
in  the  case  of  guinea  pigs,  that  horse  serum  is  poi- 
sonous to  such  animals  as  have  been  previously 
injected  with  small  amounts  of  horse  serum.2  The 
time  necessary  to  elapse  between  the  first  and  sec- 
ond injections  is  about  ten  days.  The  symptoms 
are  respiratory  embarrassment,  paralysis  and  con- 
vulsions, and  come  on  usually  within  ten  minutes 
after  the  injection.  When  death  results  it  usually 
occurs  within  one  hour,  frequently  in  less  than  thirty 
minutes.  The  poisonous  principle  in  horse  serum 
appears  to  act  on  the  respiratory  centers.  The 
heart  continues  to  beat  long  after  respiration  ceases. 
The  first  injection  of  horse  serum  renders  the 
guinea  pig  susceptible;  the  quantity  required  for 
this  purpose  is  extremely  small.  Rosenau  and 
Anderson  find  that  from  ^  to  ToW  c.c.  ordinarily 
suffice.  One  tenth  c.c.  of  horse  serum  injected  into 

1  Rosenau  and  Anderson,  Bulletin  29,  Hygienic  Laboratory, 
Washington,  1906. 

2  The  Germans  usually  speak  of  this  as  "  Theobald  Smith's 
phenomenon  of  hypersusceptibility." 


IMMUNE  SERA 

the  peritoneal  cavity  of  a  susceptible  guinea  pig  is 
sufficient  to  cause  death.  The  same  quantity  inocu- 
lated substaneously  may  cause  serious  symptoms. 
Guinea  pigs  may  be  sensitized  to  the  toxic  action  of 
horse  serum  by  feeding  them  with  horse  serum  or 
horse  meat. 

It  may  be  that  man  cannot  be  sensitized  in  the 
same  way  that  guinea  pigs  can.  However,  children 
have,  in  numerous  instances,  been  injected  with  an- 
tidiphtheric  horse  serum  at  short  and  long  inter- 
vals without,  so  far  as  we  are  aware,  causing  death. 
Certain  serums,  for  example,  the  anti tubercle  serum 
of  Maragliano  and  the  antirheumatic  serum  of  Me- 
zer,  are  habitually  used  by  giving  injections  at  inter- 
vals of  days  or  weeks.  The  results  of  Rosenau  and 
Anderson  make  it  probable  that  man  may  be  ren- 
dered sensitive  to  the  injection  of  a  strange  proteid, 
as  is  the  case  with  the  guinea  pig  and  other  animals, 
and  that  this  explanation  must  be  considered  as 
well  as  the  status  lymphaticus,  which  has  heretofore 
been  assigned  as  the  cause  of  sudden  death  following 
the  injection  of  horse  serum. 

Anaphylaxis.  —  After  the  manuscript  of  the  pres- 
ent volume  had  been  sent  to  the  printer,  a  splendid 
article  on  the  subject  of  sudden  death  in  "  sensitized 
guinea  pigs  "  made  its  appearance.  The  authors, 
Gay  and  Southard,1  have  adopted  the  term  "ana- 

1  Gay  and  Southard,  Journ.  Medical  Research,  No.  98,  May, 
1907. 


SERUM  SICKNESS  143 

phylaxis  "  for  the  phenomenon.  Their  experiments 
indicate  that  the  theory  advanced  by  v.  Pirquet  and 
Schick  is  untenable,  and  they  conclude  that  "  the 
horse  serum  contains  a  substance,  anaphylactin, 
which  is  not  absorbed  by  the  guinea  pig  tissue,  is 
not  neutralized,  and  is  eliminated  from  the  animal 
body  with  great  slowness.  When  a  normal  guinea 
pig  is  injected  with  a  small  amount  of  horse  serum, 
the  greater  part  of  its  elements  are  rapidly  elimin- 
ated ;  the  anaphylactin,  however,  remains  and  acts 
as  a  constant  irritant  to  the  body  cells,  so  that  their 
avidity  for  the  other  assimilable  elements  of  horse 
serum  which  have  accompanied  the  anaphylactin, 
becomes  enormously  increased.  At  the  end  of  two 
weeks  of  constant  stimulation  on  the  part  of  the 
anaphylactin,  and  of  constantly  increasing  avidity 
on  the  part  of  the  somatic  cells,  a  condition  has 
arrived  when  the  cells,  if  suddenly  presented  with 
a  large  amount  of  horse  serum,  are  overwhelmed 
in  the  exercise  of  their  assimilating  functions,  and 
functional  equilibrium  is  so  disturbed  that  local 
or  general  death  may  follow."  The  intoxication 
caused  by  the  second  injection  depends  upon  con- 
stituents of  the  serum  eliminable  by  the  animal 
body. 

According  to  Gay  and  Southard  the  tissue  of 
the  guinea  pig  examined  during  the  anaphylactic 
phase  showed  no  characteristic  lesions.  Striking  mul- 
tiple haemorrhages  accompany  the  toxic  phase.  The 


144  IMMUNE   SERA 

haemorrhages  are  more   frequent  in   the    stomach, 
caecum,  lungs,  and  heart  than  elsewhere. 

It  was  natural  to  think  that  a  formation  of  preci- 
pitins  was  in  some  way  responsible  for  the  symptoms 
of  serum  sickness  or  for  the  rare  cases  of  sudden 
death  following  injections  of  antitoxin  sera.  It  was 
conclusively  shown,  however,  by  v,  Pirquet  and 
Schick,  Rosenau  and  Anderson,  as  well  as  others, 
that  this  is  not  the  case.  It  was  found,  for  instance 
that  the  symptoms  of  serum  sickness  appear  within 
eight  to  thirteen  days  following  the  first  injection  of 
horse  serum,  whereas  it  requires  about  three  weeks 
for  precipitins  to  appear  in  the  blood  in  children 
after  the  injection  of  horse  serum.  Furthermore, 
the  formation  of  precipitins  does  not  take  place  as 
readily  in  man  following  the  injection  of  horse  serum 
as  it  does  in  rabbits.  In  fact,  v.  Pirquet  and 
Schick  found  that  sometimes  even  after  the  injec- 
tion of  200  c.c.  there  was  no  production  of  precip- 
itins. Finally  Rostoski  has  called  attention  to  the 
fact  that  the  precipitin  action  is  a  test-tube  pheno- 
menon only,  and  does  not  occur  in  vivo.  It  is  well 
to  bear  these  facts  in  mind.  In  a  recent  discussion 
on  the  treatment  of  severe  cases  of  diphtheria  in 
which  the  intravenous  administration  of  large  doses 
of  antitoxin  was  recommended,  one  of  the  speakers 
alluded  to  the  dangers  from  precipitin  formation  as 
contra  indicating  such  a  procedure.  Such  fears  are 
groundless. 


SERUM   SICKNESS  145 

The  Concentration  and  Purification  of  Antitoxic 
Sera.  —  Since  the  development  of  serum  rashes  and 
other  disagreeable  symptoms  is  largely  associated 
with  the  serum  as  serum,  it  was  natural  that  at- 
tempts should  be  made  to  concentrate  the  serum  as 
much  as  possible.  This  was  sought  to  be  accom- 
plished in  two  ways,  —  (i )  by  causing  the  production 
by  the  horses  of  a  high  grade  serum,  (2 )  by  separating 
the  non-antitoxic  from  the  antitoxic  fractions  of  the 
serum.  Without  going  into  details,  we  may  say 
that  the  average  grade  of  antitoxin  at  present  pro- 
duced is  from  five  to  ten  times  stronger  than  the 
early  Behring  sera.  We  have,  however,  in  that 
time  also  markedly  increased  the  number  of  units 
ordinarily  given  per  dose,  so  that  the  volume  of 
serum  is  still  considerable.  So  far  as  the  separa- 
tion of  the  antitoxic  and  non-antitoxic  fraction  is 
concerned  we  have  already  referred  to  the  great 
advance  made  by  Gibson  l  in  the  practical  concen- 
tration and  purification  of  diphtheria  antitoxin. 
It  remains  here  to  consider  what  clinical  results 
have  been  achieved  with  this  globulin  preparation. 
In  a  recent  study  of  this  question  Park  and  Throne  2 
conclude  that  "  the  removal  of  a  considerable 
portion  of  the  non-antitoxic  globulins,  as  well  as 
all  the  albumins  from  the  serum  by  the  Gibson 
method  has  eliminated  much  of  the  deleterious 

1  See  page  18. 

*  Park  and  Throne,  Am,  Journ,  of  the  Med.  Sciences,  1906. 


146  IMMUNE    SERA 

matter  from  the  serum,  so  that  severe  rashes,  joint 
complications,  fever,  and  other  constitutional  dis- 
turbances are  less  likely  to  occur  from  the  antitoxic 
globulins  than  from  the  antitoxic  serum  from  which 
it  was  obtained."  Similar  favorable  reports  have 
been  published  by  other  observers. 


APPENDIX  A. 

THE    WASSERMANN   TEST    FOR    SYPHILIS. 

As  has  already  been  pointed  out  on  page  69, 
Wassermann  applied  the  principle  of  the  Bordet- 
Gengou  phenomenon  to  the  detection  of  syphilis 
antibodies  in  the  serum  and  cerebrospinal  fluid  of 
persons  infected  with  syphilis.  In  the  two  years 
which  have  elapsed  since  Wassermann's  first  pub- 
lication, the  reliability  of  this  method  of  diagnos- 
ing syphilis  has  been  confirmed  by  a  large  number 
of  investigators,  and  it  has  already  proven  of  con- 
siderable value  in  several  departments  of  medicine. 
In  response  to  numerous  requests,  the  writer  has 
undertaken  to  give  a  clear  description  of  the  test, 
together  with  a  brief  review  of  the  results  thus  far 
achieved  by  its  use. 

When  an  animal  is  repeatedly  injected  with  red 
blood  cells  of  another  species,  it  reacts  to  such  in- 
jections by  producing  substances  in  its  serum  which 
have  the  power  to  dissolve  these  foreign  blood  cells. 
Examined  by  means  of  a  test  tube  experiment,  it 
is  found  that  the  serum  exerts  this  power  only 
while  it  is  fresh.  Serum  several  days  old  is  unable 
to  dissolve  the  red  cells.  The  fresh  serum  also 

147 


J48  APPENDIX. 

loses  its  solvent  power  by  exposure  to  heat,  say  to 
55°  C.  Investigations  showed  that  the  solvent 
action  could  be  restored  to  these  sera  by  the  addi- 
tion of  small  quantities  of  a  fresh  normal  serum, 
i.e.  of  a  serum  which  by  itself  had  no  solvent  power 
whatever.  The  inactive  serum  had  thus  been 
reactivated.  The  original  specific  dissolving  serum 
therefore  contained  two  substances,  one  of  which 
is  very  labile  and  the  other  stable.  The  stable 
substance  is  specific  for  the  blood  cells  against 
which  it  is  directed,  i.e.  against  the  cells  used  for 
immunizing  the  animal.  It  is  called  the  "  immune 
body,"  or  the  "amboceptor."  The  labile  sub- 
stance, as  we  have  seen,  is  present  in  all  fresh  sera, 
and  is  spoken  of  as  the  " complement."  The  action 
of  the  immune  body  seems  to  consist  in  bringing 
the  solvent  action  of  the  complement  to  bear  on 
the  given  cells.  We  must  conceive  that  the  com- 
plement possesses  the  solvent  power,  but  has  no 
way  of  laying  hold  of  the  cell  to  be  dissolved.  The 
immune  body  merely  effects  this  combination. 
Ehrlich's  diagram  on  page  64  will  serve  to  make 
this  conception  clear. 

All  that  has  been  said  regarding  immune  bodies 
and  complement  for  the  solution  of  blood  cells, 
holds  for  the  substances  which  effect  destruction  of 
bacteria  when  bacteria  are  used  for  immunization. 
In  fact,  the  process  is  the  same,  no  matter  what 
cells  are  injected  into  the  animal.  The  immune 
body  is  always  directed  specifically  against  the 
cells  injected,  and  against  no  others. 


APPENDIX. 


149 


As  can  be  seen  from  Ehrlich's  diagrams,  the 
bacteria  or  blood  cells  combine  directly  only  with 
the  immune  body.  The  complement,  as  already 
said,  has  no  way  of  laying  hold  of  the  cells.  As 
soon  as  the  bacteria  or  cells  have  anchored  the 
immune  body,  however,  conditions  change.  The 
combination  at  once  attracts  and  unites  with  the 
complement.  If  the  amount  of  complement  is  not 
too  large,  the  combination  may  unite  with  all  of  it, 
i.e.  may  abstract  the  complement  from  the  serum. 

Just  let  us  examine  this  by  means  of  an  illustra- 
tion :  Let  us  suppose  we  have  immunized  an  ani- 
mal with  typhoid  bacilli,  and  have  obtained  a 
specific  serum  directed  against  these  bacilli.  This 
serum  has  been  inactivated  by  heating  it  to  55°  C., 
so  that  now  it  will  act  on  typhoid  bacilli  only  when 
some  fresh  normal  serum  is  added  to  complement 
the  immune  body.  For  this  purpose  we  have 
provided  ourselves  with  some  freshly  drawn  serum 
from  a  guinea  pig.  The  guinea  pig  serum,  there- 
fore, is  the  " complement."  On  mixing  typhoid 
bacilli  with  the  specific  immune  serum  and  then 
with  the  complement,  these  three  factors  enter  into 
combination,  and  this  results  in  the  destruction  of 
the  typhoid  bacilli.  The  quantities  can  easily  be 
so  arranged  that  this  combination  uses  up  all  of  the 
complement,  so  that  the  fluid  contains  not  a  trace 
of  free  complement  after  the  substances  have  com- 
bined. 

Suppose,  now,  that  we  also  had  a  specific  serum 
obtained  by  injecting  an  animal  with  red  blood 


150  APPENDIX. 

cells,  for  example,  by  injecting  a  rabbit  with  sheep 
blood  cells.  This  rabbit  serum  would  then  be 
specifically  directed  against  sheep  blood  cells.  Let 
us  inactivate  this  serum,  by  heating  it  to  55°  C.,  so 
that  now  it  requires  the  addition  of  a  fresh  normal 
serum  to  exert  its  solvent  effect.  For  this  purpose 
we  can  again  use  fresh,  normal  guinea-pig  serum. 
When,  then,  we  mix  sheep  blood  cells  with  our 
specific  immune  serum  (against  sheep  blood  cells) 
and  with  the  complement,  i.e.,  with  fresh  normal 
guinea-pig  serum,  all  three  factors  unite,  and  bring 
about  destruction  of  the  red  blood  cells.  This  is 
manifested  by  the  blood  cells  dissolving  and 
giving  off  their  haemoglobin  to  the  rest  of  the 
fluid. 

Let  us  now  suppose  we  have  carried  out  the  first 
part  of  this  experiment,  that  with  the  typhoid  bacilli, 
and  have  left  typhoid  bacilli,  specific  typhoid  serum 
and  complement  in  contact  for  several  hours  in  a 
warm  place  in  order  to  cause  the  three  factors  to 
combine.  At  the  end  of  this  time  let  us  add  sheep 
blood  cells  and  the  specific  serum  directed  against 
sheep  cells,  but  let  us  add  no  further  complement, 
because  the  fresh  guinea-pig  serum  was  able,  as  we 
saw,  to  serve  as  as  complement  also  for  the  blood 
combination.  The  mixture  is  again  placed  in  a 
warm  place  for  several  hours,  and  then  for  twenty- 
four  hours  in  the  refrigerator,  after  which  it  is  ex- 
amined. We  shall  find  that  no  haemolysis  has 
occurred,  from  which  we  conclude  that  the  previous 
combination  (typhoid  bacilli,  immune  scrum  and 


APPENDIX.  151 

complement),  had  used  up  all  the  complement,  and 
left  none  for  the  blood  combination. 

If  we  were  to  repeat  the  whole  experiment,  but 
leave  out,  in  the  first  part  of  the  test,  say  the  spe- 
cific typhoid  serum,  we  should  find  that  the  blood 
cells  would  be  dissolved.  This  is  readily  under- 
stood when  it  is  remembered  that  then  we  would 
have  only  typhoid  bacilli  and  complement,  two 
factors  which  cannot  ccmbine  directly.  The  com- 
plement would  therefore  be  left  free  to  act  in  the 
blood  combination. 

If  hasmolysis  occurs  we  may  therefore  conclude 
that  one  of  the  factors  in  the  first  combination  was 
absent,  and  conversely,  if  haemolysis  does  not  occur, 
we  know  that  the  first  combination  must  have 
been  perfect,  i.e.  all  three  factors  must  have  been 
present. 

It  is  at  once  apparent  that  in  adapting  this  test 
to  the  detection  of  syphilis  antibodies,  pure  cultures 
of  the  causative  organism,  i.e.  of  the  "  antigen," 
were  not  available.  Wassermann  therefore  made 
use  of  extracts  of  syphilitic  organs  rich  in  spiro- 
chaetes  in  place  of  the  typhoid  bacilli,  and  used 
either  the  serum  or  the  cerebrospinal  fluid  of  the 
suspected  case  in  place  of  the  typhoid  antiserum. 
The  rest  of  the  test  was  similar  to  that  described 
above.  When  hasmolysis  of  the  sheep  cells  occured, 
Wassermann  said  it  showed  that  the  first  combina- 
tion was  incomplete;  when  haemolysis  was  com- 
pletely inhibited,  it  showed,  he  said,  that  the  first 
combination  was  perfect ;  i.e.  that  the  serum  or 


152 


APPENDIX. 


spinal  fluid  contained  syphilis  antibody.  As  in 
most  such  tests,  only  a  positive  result  determines ; 
a  negative  result  does  not  necessarily  exclude  the 
presence  of  syphilitic  infection. 

While  the  above  exposition  will  serve  to  fix  the 
general  plan  of  the  test  in  the  mind  of  the  reader, 
we  must  at  once  say  that  the  mode  of  action  is  not 
as  simple  as  Wassermann  first  believed.  Before 
going  into  this  phase  of  the  subject,  it  will  be 
advisable  to  present  a  description  of  the  technique 
of  the  test. 

For  carrying  out  the  test  the  following  materials 
are  required: 

(1)  Antigen,    i.e.    fluid   containing   syphilis   ma- 
terial.     This  is  comparable  to  the  pure  culture  of 
typhoid  in  the  test  described  above. 

(2)  Serum  or  cerebrospinal  fluid  from  the  patient 
to  be  examined. 

(3)  Sheep  blood  cells. 

(4)  Hasmolytic  antibody,  i.e.    inactivated  serum 
of  a  rabbit  immunized  against  sheep  blood  cells. 

(5)  Complement,  i.e.  fresh  normal  serum  from  a 
guinea-pig. 

For  the  syphilis  antigen  it  is  best  to  use  the 
organs  of  a  syphilitic  fcetus,  i.e.  one  dead  of  heredi- 
tary syphilis,  as  these  tissues  are  particularly  rich 
in  spirochaetes.  The  organs  are  chopped  up  and 
macerated  in  a  clean  vessel  in  a  mixture  composed 
of  water,  1000;  NaCl,  8.5;  carbolic  acid,  5.0;  one 
part  of  the  tissue  to  four  of  the  fluid.  The  mixture 
is  shaken  in  a  shaking  apparatus  for  twenty  hours ; 


APPENDIX. 


153 


the  supernatant  fluid  poured  off  and  centrifuged  so 
as  to  be  perfectly  clear.* 

The  serum  for  the  test  is  collected  from  the 
patient  in  the  usual  way  by  drawing  from  5  to 
10  cc.  of  blood  from  a  vein  at  the  elbow,  placing 
the  blood  in  a  sterile  test  tube  and  allowing  it  to 
clot.  Cerebrospinal  fluid,  obtained  by  lumbar 
puncture,  is  preserved  with  0.5%  carbolic  acid,  and 
then  strongly  centrifuged  so  as  to  make  it  perfectly 
clear. 

The  sheep  blood  cells  are  obtained  by  defibrinat- 
ing  sheep  blood,  centrifuging  and  washing  the  blood 
cells  repeatedly  with  normal  salt  solution  to  remove 
traces  of  adherent  serum.  A  5%  suspension  in  salt 
solution  is  used. 

The  hasmolytic  antibody  consists  of  the  serum  of 
a  highly  immunized  (against  sheep  blood  cells) 
rabbit,  the  serum  being  inactivated  by  heating  to 
56°  C.  In  the  tests  cited  by  Wassermann,  one  cc. 
of  a  1/1500  dilution  of  serum  dissolved  one  cc.  of 
5%  suspension  of  sheep  blood  cells  at  37°  C.  in 
two  hours. 

The  complement  consists  of  freshly  drawn  guinea- 
pig  serum.  The  test  is  carried  out  as  follows : 

To  constant  quantities  of  spinal  fluid  (e.g.  i  cc. 
of  the  i/io  dilution)  decreasing  amounts  of  the 
extract  of  organs  are  added,  thus  0.2,  o.i,  0.05  cc. 
Then  i  cc.  of  a  i/io  dilution  of  fresh  normal  guinea- 

*  In  a  very  recent  article,  Wassermann  states  that  more  uni- 
formly active  extracts  can  be  obtained  by  using  96%  alcohol  in 
place  of  the  water  in  the  above  formula. 


154  APPENDIX. 

pig  serum  is  added,  and  the  mixtures  allowed  to 
remain  in  contact  at  37°  C.  for  one  hour  in  order  to 
bind  the  complement. 

In  this  mixture  we  have  antigen;  wre  may  or  may 
not  have  antibody;  we  have  complement. 

If  the  antibody  is  present,  the  complement  will 
be  anchored  by  the  combination,  and  so  be  unavail- 
able for  the  hasmolytic  test  next  in  order.  If  no 
antibody  is  present,  the  complement  will  still  be 
free  to  act  in  the  hasmolytic  test. 

At  the  end  of  the  hour,  we  add  to  the  above 
mixtures:  one  cc.  of  a  5%  suspension  of  sheep 
blood  cells,  and  one  cc.  of  the  amboceptor  dilution 
containing  double  the  solvent  dose  for  that  amount 
of  sheep  blood  cells.  Thus,  if  the  titer  of  the 
haemolytic  serum  is  1/1800,  we  take  one  cc.  of  a 
dilution  1/900. 

All  the  tubes  are  made  up  to  the  same  volume 
with  normal  salt  solution,  namely,  to  5  cc.,and  are 
then  placed  in  the  incubator  at  37°  C.  and  kept 
there  for  two  hours.  Then  they  are  placed  on  ice 
until  the  next  day,  when  the  results  are  noted1 
The  whole  procedure  is  clearly  shown  by  the  pro- 
tocol from  Wassermann  and  Plaut  reproduced  0:1 
page  155. 

Few  experiments  in  immunity  require  such  care- 
ful technique,  or  are  open  to  so  many  sources  of 
error  as  this  serum  test  for  syphilis.  In  view, 
too,  of  the  enormous  responsibility  assumed  in 
making  a  positive  diagnosis  of  syphilis,  it  is  appa- 
rent that  the  test  should  only  be  undertaken  by 


APPENDIX. 


155 


Hsemolv- 

tic  Ambo- 

Svphilit 
Foetus 
Extract. 
(0.2  gm.) 
One  c.c. 
of  ^  Dilu- 
tion. 

Spinal  Fluid 
of  Patient 
M.  0.2.  i.e., 
One  c.o.  of 
the  5  Dilu- 
tion. 

Normal 
Guinea- 
pig  Serum 

O.  I    CO.. 

i.e.  i  cc. 
of  a  T\j 
Dilution. 

ceptor 

I    CC. 

equals 
Double 
the 
Solvent 
dose  for 

Sheep 
Blood 
Cells  i  cc. 
of  a  5% 
Suspen- 
sion. 

Results. 

i  cc.  of  a 

5%  Sus- 

pension. 

O.  2 

O.  2 

I.O 

I.O 

I.O 

Complete  inhibition 

of  haemolysis 

O.I 

0.2 

I.O 

I.O 

i  .0 

Compl.  inhibition 

0.2 

O.I 

I.O 

•I.O 

I.O 

Marked  inhibition 

O.I 

O.  I 

I.O 

I.O 

I.O 

Marked  inhibition 

0.2 

I.O 

I.O 

I.O 

Complete  solution 

O.I 

I.O 

I.O 

I.O 

Complete  solution 



0.  2 

I.O 

1  .0 

1  .0 

Complete  solution 



0.  I 

I.O 

I.O 

I.O 

Complete  solution 

Spinal  Fluid 

of 

Non-syphil. 

Person. 

O.  2 

0.2 

I.O 

I.O 

I.O 

Complete  solution 

0.  I 

O.  2 

I.O 

I.O 

I.O 

Complete  solution 

0.2 

O.  I 

1  .0 

1  .0 

I.O 

Complete  solution 

O.I 

O.I 

I.O 

I.O 

I.O 

Complete  solution 



0.2 

I.O 

I.O 

I.O 

Complete  solution 

O.I 

I.O 

I.O 

1  .0 

Complete  solution 

highly  trained  laboratory  workers.  On  the  other 
hand,  most  who  have  busied  themselves  with  the 
test  agree  that  suitable  controls  always  lead  to  a 
detection  of  possible  sources  of  error,  and  that 


156  APPENDIX. 

therefore  the  reaction,  when  properly  performed, 
can  be  relied  upon. 

When  the  test  was  first  published  Wassermann 
regarded  the  reaction  which  occurred  as  one  between 
mutually  specific  bodies,  i.e.  between  antigen  and 
antibody,  the  resulting  combination  having  the 
power  to  anchor  the  complement.  Through  the 
work  of  Marie  &  Levaditi,  of  Landsteiner,  Miiller 
and  Potzl,  of  Weil  and  Braun,  and  still  other  in- 
vestigators, it  soon  became  apparent  that  the  test 
could  also  be  carried  out  by  using  extracts  of  non- 
syphilitic  tissue,  i.e.  of  other  pathological  tissue  or 
normal  tissue.  That,  of  course,  meant  that  the 
view  of  a  reciprocal  specific  relation  between  anti- 
body and  organ  extract,  in  the  sense  that  typhoid 
antibody  and  typhoid  bacilli  are  reciprocally  re- 
lated, had  to  be  abandoned.  This  does  not,  how- 
ever, effect  the  reliability  of  the  reaction  for  diag- 
nostic purposes,  for  it  has  been  found  that  positive 
results  are  still  only  obtained  when  the  serum  or 
spinal  fluid  is  of  syphilitic  origin.* 

Working  under  Wassermann's  direction,  Forges 
and  Meier  stu.died  the  nature  of  the  substances 
concerned  in  the  reaction,  and  began  by  precipi- 
tating the  organ  extracts  with  alcohol  and  testing 

*  It  may  be  well  to  state  that  according  to  Landsteiner,  Muller 
and  Potzl  the  serum  of  animals  infected  with  dourine  (trypanoso- 
miasis)  also  gives  rise  to  inhibition  of  haemolysis  when  tested 
according  to  the  above  method.  This  has  been  confirmed  by 
Hartoch  and  Yakimoff.  Whether  this  will  affect  the  value  of  the 
Wassermann  test  in  humans  can  only  be  decided  by  further 
clinical  tests,  especially  in  cases  of  human  trypanosomiasis. 


APPENDIX.  !57 

the  resulting  precipitate  and  clear  fluid  separately. 
It  was  found  that  the  substance  concerned  in  the 
reaction  was  soluble  in  alcohol,-  and  the  authors 
thereupon  made  alcoholic  extracts  of  the  syphilitic 
organs.  These  worked  satisfactorily  in  making  the 
test.  It  was  natural  to  think  that  the  substance 
which  effected  the  reaction  might  be  related  to  the 
lipoids,  and  so  the  authors  next  studied  the  be- 
havior of  alcoholic  extracts  of  normal  human  and 
animal  organs.  While  these  extracts  also  sufficed 
to  produce  the  reaction,  it  was  evident  that  they 
were  not  as  active  as  extracts  from  syphilitic 
organs.  After  it  had  been  found  that  alcoholic  ex- 
tracts could  be  used  for  the  test,  a  number  of 
authors  almost  simultaneously  published  favorable 
results  with  chemically  defined  substances.  Forges 
and  Meier  used  lecithin,  Levaditi  glycocholate  of 
soda,  Sachs  and  Altmann  oleate  of  soda,  and 
Fleischmann  even  used  vaseline.  The  last-named 
also  used  cholesterin  with  favorable  results,  but 
Forges  and  Meier  obtained  only  negative  results 
with  this  substance.  On  the  whole,  however,  it 
seems  that  the  extracts,  especially  of  syphilitic 
organs,  give  the  most  uniform  results. 

At  the  present  time,  therefore,  Wassermann  be- 
lieves that  the  really  active  principle  in  the  antigen 
may  be  a  combination  of  lipoids  with  certain  protein- 
like  substances,  and  that  the  latter  component, 
when  it  is  derived  from  syphilitic  material,  has 
something  of  a  specific  character.  In  this  connec- 
tion Wassermann  refers  to  the  researches  of 


158  APPENDIX. 

Noguchi,  Landsteiner,  and  others  which  show  that 
minute  quantities  of  proteid  mixed  with  lipoids 
may  cause  extensive  alterations  in  the  physico- 
chemical  behavior  of  the  latter.  He  thinks  that 
under  certain  circumstances  this  proteid  component 
may  play  an  important  role  in  determining  the 
reliability  of  the  reaction,  a  view  which  is  borne  out 
by  the  investigations  of  Neisser  and  Bruck. 

While  Forges  and  Meier  were  engaged  in  the 
studies  just  mentioned,  Fornet  and  also  Michaelis 
showed  that  when  the  serum  of  individuals  infected 
with  syphilis  was  mixed  with  certain  antigens  a 
zone  of  precipitation  might  at  times  be  observed  at 
the  point  of  contact  of  the  two  fluids.  The  antigen 
employed  by  Fornet  was  serum  from  individuals  in 
the  florid  stage  of  syphilis ;  Michaelis  used  extracts 
of  organs  from  a  syphilitic  foetus.  This  of  course 
agrees  with  what  was  already  known  from  the  work 
of  Bordet,  Gengou,  and  Gay.  In  fact,  according 
to  Gay,  the  deflection  or  absorption  of  complement, 
on  which  the  Bordet-Gengou  test  depends,  may  be 
due  to  the  precipitate  formed  in  the  combination. 
While  this  is  true,  complement  may  also  be  anchored 
in  the  Bordet-Gengou  test  without  the  formation  of 
any  precipitate. 

Forges  and  Meier  thereupon  tested  the  alcoholic 
extracts,  and  solutions  of  lecithin  and  of  glycocho- 
late  of  soda  to  see  whether  this  zone  of  precipita- 
tion was  at  all  constant,  and  whether  it  might  not 
be  possible  to  substitute  such  a  simple  precipitation 
test  for  the  complicated  Wassermann  reaction. 


APPENDIX.  159 

While  it  was  found  that  the  test  was  roughly  spe- 
cific, it  was  soon  realized  that  a  precipitate  might 
at  times  be  produced  with  the  serum  of  surely 
non-syphilitic  individuals,  and  similar  unfavorable 
result^  have  since  been  published  by  other  authors. 
At  the  present  time,  therefore,  the  only  reliable 
serum  diagnosis  of  syphilis  is  that  based  on  the 
absorption  of  complement. 

The  results  obtained  with  the  Wassermann  test  are 
well  reflected  in  the  findings  of  Fleischmann,  as 
follows : 

The  total  number  of  persons  tested  was  230,  of 
which  38  were  controls.  None  of  the  latter  gave  a 
positive  reaction.  The  other  cases  can  be  arranged 
into  four  groups  thus : 

1 i )  Cases  surely  syphilitic,  with  clinically  manifest 
signs  of  syphilis  at  the  time  of  the  test.    Of  89  such 
cases  tested,  83  gave  a   positive  reaction  (93%). 

(2)  Cases    surely  syphilitic    but   without  clinical 
symptoms    at    the  time   of  the  test.      Of  64  such 
cases  tested,  33  gave  a  positive  reaction  (52%),  and 
31  gave  a  negative  reaction  (48%). 

(3)  Cases  with  symptoms  suggestive  of  syphilis, 
and  with  an  indefinite  history  of  infection.     Of  32 
such  cases,  16  gave  a  positive  reaction  (50%),  and 
the  rest  a  negative  reaction. 

(4)  Surely  syphilitic  individuals  showing  cutaneous 
lesions  which  the  dermatologists  diagnosed  as  very 
probably  not  syphilitic.      Of  7  such  cases,  i  gave  a 
positive  reaction  and  the  rest  a  negative  reaction. 

Bruck  and  Stern  tested  378  cases  suspected  to  be 


l6o  APPENDIX. 

syphilitic,  and  obtained  a  positive  reaction  in  204. 
They  also  tested  157  surely  non-syphilitic  individuals 
as  controls,  and  found  all  but  two  negative.  These 
two  gave  a  doubtful  reaction. 

In  a  recent  paper  Wassermann  has  collected  data 
on  about  3000  tests,  as  follows:  There  were  1010 
tests  on  cases  surely  non-syphilitic  (controls),  and 
not  one  of  these  gave  a  positive  reaction.  Of  the 
1982  surely  syphilitic  cases  tested,  those  examined 
at  the  time  when  they  had  manifest  symptoms 
reacted  in  about  90%  of  the  cases.  When  the  cases 
tested  were  without  manifest  symptoms,  so-called 
" latent  syphilitics,"  about  50%  reacted. 

As  a  matter  of  interest  it  may  be  mentioned  that 
Blumenthal  and  Wile  tested  the  urine  of  syphilitic 
individuals,  and  found  that  this  too  would  give  the 
reaction. 

Marie  and  Levaditi  examined  the  cerebrospinal 
fluid  of  30  cases  of  general  paresis.  All  but  two  of 
the  cases  gave  a  positive  reaction.  When  the  serum 
was  tested  in  place  of  the  cerebrospinal  fluid,  the 
percentage  of  positive  findings  dropped  to  59%. 

Michaelis  examined  20  cases  of  general  paresis 
and  obtained  a  positive  reaction  in  1 8  of  them. 

Citron  examined  43  tabetics  and  paretics,  and  ob- 
tained a  positive  reaction  in  34  cases  (79%).  He 
also  tested  the  serum  of  108  persons  surely  infected 
with  syphilis,  or  suspected  to  be  infected,  and  ob- 
tained a  positive  reaction  in  80  (74%).  None  of  the 
sera  from  156  surely  non-syphilitic  individuals  gave 
a  positive  reaction. 


APPENDIX.  161 

Favorable  reports  have  also  been  published  con- 
cerning the  reliability  of  the  test  in  ophthalmology, 
dermatology,  and  other  departments  of  medicine. 

At  the  present  time  we  may  therefore  say  that  the 
chief  value  of  the  serum  test  for  syphilis  will  be  in 
those  cases  in  which  there  are  symptoms  suggestive 
of  syphilis  and  in  which  the  history  of  the  case  fails 
us  or  is  of  questionable  reliability.  In  such  in- 
stances a  positive  reaction  at  once  clears  the  diag- 
nosis, and  sometimes  even  a  negative  reaction  is 
helpful. 

The  following  are  some  of  the  more  important 
references  to  the  subject: 

BORDET  and  GENGOU.     Annales  Pasteur,  Vol.  XV.     1901. 

GAY.     Centralblatt  Bacteriologie,  Originale,  Vol.  39.     1905. 

WASSERMANN,  NEISSER,  and  BRUCK.  Deutsche  med.  Wochen- 
schrift,  No.  19.  1906. 

WASSERMAN  and  PLAUT.     Ibid.  No.  44.     1906, 

NEISSER,  BRUCK,  and  SCHUCHT.     Ibid.  No.  48.     1906. 

LANDSTEINER  and  STANKOVIC.  Centralblatt  Bacteriologie, 
Originale,  Vol.  42.  1906. 

MARIE  and  LEVADITI.     Annales  Pasteur,  Vol.  21.     1907. 

LANDSTEINER,  MULLER,  and  POTZL.  Wiener  klinische 
Wochenschrift,  No.  17.  1907. 

FORNET  and  SCHERESCHEWSKI.  Miinchener  med.  Wochen- 
schrift, No.  30.  1907. 

CITRON.     Berliner  klinische  Wochenschrift,  No.  43.     1907. 

MICHAELIS.     Ibid.  No.  46.     1907. 

WEIL  and  BRAUN.     Ibid.  No.  49.     1907. 

WASSERMANN.     Ibid.  No.  50  and  No.  51.     1907. 

PORGES.     Ibid.  No.  51.     1907. 


1 62  APPENDIX. 

FISCHER  and  MEIER.     Deutsche  medizinische  Wochenschrif t , 

page  2169.    1907. 
LEVADITI   and    YAMANOUCHI.     Comptes   rendus   societe   de 

Biologic,  Vol.  63.     1907. 
LEVADITI.     Presse  medicale,  No.  90.     1907. 
FLEISCHMANN  and  BUTLER.     Journal  Americ.  Medical  Ass'n. 

Sept.  14,  1907. 

KAREWSKI.    Berliner  klinische  Wochenschrift,  No.  i.     1908. 
MICHAELIS  and  LESSER.     Ibid.  No.  6.     1908. 
KRONER.    Ibid,  page  149.     1908. 
FORNET   and    SCHERESCHEWSKI.      Munchener   medizinische 

Wochenschrift,  No.  6.     i9o8. 
PLAUT,  HEUCK,  and  Rossi.     Ibid,  page  66.     1908. 
CITRON.     Berliner  klinische  Wochenshrift,  No.  10.     1908. 
MEIER.     Ibid.  No.  10,  1908. 
SACHS  and  ALTMANN.     Ibid.  No.  10.     1908. 
FLEISCHMANN.    Ibid.  No.  10.     1908. 

ELIAS,  NEUBAUER,  and  PORGES.    Wiener  klinische  Wochen- 
schrift, No.  ii.     1908. 
SACHS  and  ALTMANN.     Berliner  klinische  Wochenschrift,  No. 

14.     1908. 

PORGES  and  MEIER.     Ibid.  No.  15.     1908. 
FRITZ  and  KREN.     Wiener  klinische  Wochenschrift,  No.  12. 

1908. 
WASSERMANN.     Munchener  medizinische  Wochenschrift,  No. 

17.     1908. 

PORGES.     Ibid.  No.  17.     1908. 
v.    EISLER.       Wiener     klinische    Wochenschrift,     No.     13. 

1908. 

WEIL  and  BRAUN.  Ibid.  No.  17.  1908. 
GROSZ  and  VOLK.  Ibid.  No.  18.  1908. 
ELIAS,  NEUBAUER,  PORGES,  and  SALOMON.  Ibid.  No.  18. 

1908. 


APPENDIX.  163 

OPPENHEIM.     Ibid.  No.  19.     1908. 

HARTOCH  and  YAKIMOFF.     Ibid.  No.  21.     1908. 

ELIAS,  NEUBAUER,  FORGES  and  SALOMON.      Ibid.  No.   21. 

1908. 

WASSERMANN.     Ibid.  No.  21.     1908. 
BLUMENTHAL  and  U.  J.  WILE.     Berliner  klinische  Wochen- 

schrift,  No.  22.     1908. 


APPENDIX   B. 

NOGUCHI'S    BUTYRIC   ACID   TEST. 

FEELING  that  the  lecithin,  glycocholate  of  soda, 
oleate  of  soda,  and  other  compounds  which  had 
been  used  in  the  various  modifications  of  the 
Wassermann  test,  might  act  as  acids,  and  that  this 
acidification  possibly  gave  rise  to  a  precipitate  of 
globulins  and  related  substances,  Noguchi  sought 
to  discover  the  nature  of  the  Wassermann  reaction 
by  studying  the  influence  of  various  acids  on  the 
serum  of  different  individuals.  He  found  that  in 
general  a  greater  degree  of  precipitation  was  pro- 
duced in  syphilitic  sera  than  in  non-syphilitic  ones. 
It  was  natural  to  think  that  the  deflection  of  com- 
plement observed  in  the  Wassermann  test  was  due 
to  adsorption  by  the  precipitate  as  such,  but  on 
testing  the  precipitate  itself  this  was  found  not  to 
be  the  case. 

The  increased  precipitation  produced  in  syphilitic 
sera  by  the  addition  of  acid  suggested  an  increase 
and  qualitative  change  on  the  part  of  the  serum 
globulin,  and  this  possibility  was  also  indicated  by 
the  results  obtained  by  Klausner.  This  author,  it 
may  be  stated,  devised  a  test  for  syphilis,  based 
on  the  formation  of  a  precipitate  when  the  serum 

164 


APPENDIX  165 

was  mixed  with  distilled  water.  Noguchi  then 
made  exact  determinations  of  the  globulin  content 
of  the  different  sera,  and  found  that  in  cases  of 
secondary  syphilis  either  untreated  or  but  slightly 
treated,  an  increased  globulin  content  could  be 
demonstrated.  In  primary  and  tertiary  stages  the 
change  was  found  to  be  inconstant.  The  results 
of  these  globulin  determinations  were  quite  gen- 
erally paralleled  by  the  result  of  the  Wassermann 
test  on  the  sera  question.  The  method  employed 
for  determining  the  globulin  content  of  the  serum 
was  the  ordinary  one  of  the  biochemical  laboratory, 
as  follows : 

The  serum  was  mixed  with  half -saturated  solu- 
tion of  ammonium  sulphate  and  the  resulting  pre- 
cipitate concentrated  always  to  the  same  volume 
by  means  of  a  centrifuge.  After  pouring  off  the 
supernatant  fluid,  the  precipitate  was  carefully 
weighed  in  its  moist  condition. 

It  is  evident  that  this  method  can  be  employed 
only  by  trained  workers  in  suitable  laboratories: 
it  is  hardly  clinically  applicable.  In  order  to  over- 
come this  objection,  Noguchi  devised  the  following 
simple  modification: 

The  ammonium  sulphate  precipitate  is  separated 
by  contrifuging  as  before,  and  the  supernatant 
fluid  poured  off.  The  precipitate  is  then  redis- 
solved  by  adding  ten  volumes  of  physiological  salt 
solution,  and  tested  by  the  addition  of  a  10%  solu- 
tion of  butyric  acid  in  salt  solution.  When  the 
globulin  content  o:  the  serum  is  normal  slight 


1 66  APPENDIX. 

opalescence  is  produced,  but  with  an  increased 
globulin  content,  such  as  is  seen  in  secondary 
syphilis,  the  mixture  becomes  cloudy  and  shows 
a  distinct  flocculent  precipitate  in  about  half  an 
hour.  With  butyric  acid  the  appearance  of  the 
precipitate  is  quite  characteristic;  with  other  acids 
much  less  differentation  is  obtained. 

The  technique  of  the  butyric  acid  test  is  as 
follows : 

For  Serum — To  i  cc.  serum  add  4  cc.  half-satu- 
rated solution  ammonium  sulphate.  After  two 
hours,  centrifuge  at  high  speed  for  15  minutes. 
Pour  off  the  supernatant  fluid,  and  dissolve  the 
precipitate  in  10  cc.  physiological  salt  solution. 
To  0.5  cc.  of  this  solution  add  0.5  cc.  of  a  10% 
solution  of  butyric  acid  in  salt  solution.  A  floccu- 
lent precipitate  within  two  hours  constitutes  a 
positive  test.  Readings  made  after  that  time  may 
lead  to  erroneous  conclusions,  as  even  non-syphili- 
tic sera  may  give  a  slight  precipitation  under  these 
circumstances. 

For  Cerebro >  spinal  Fluid. — The  preliminary  pre- 
cipitation with  ammonium  sulphate  is  omitted. 
To  o.i  cc.  of  the  fluid  add  0.5  cc.  of  the  10%  solu- 
tion of  butyric  acid  in  salt  solution.  Heat  to  boil- 
ing, and  add  o.i  cc.  normal  NaOH.  Observe  the 
tubss  at  the  end  of  ten  to  twenty  minutes.  A  posi- 
tive reaction  is  indicated  by  the  appearance  of  a 
coarsely  granular  or  flocculent  precipitate.  With 
a  negative  reaction  there  is  merely  a  uniform  cloud- 
ing, but  no  such  precipitate. 


APPENDIX. 


I67 


Noguchi's  results  with  the  butyric  acid  test  are 
shown  in  the  following  summary : 

(1)  The  cerebrospinal  fluid  of  40  cases  of  general 
paresis  was   tested.      40    gave  a  positive   reaction 
with   Noguchi's   test;    34  a  positive  reaction  with 
Wassermann's  test. 

In  all  the  cases  where  a  positive  reaction  was 
obtained,  the  diagnosis  agreed  with  that  based  on 
the  cytological  and  clinical  findings. 

(2)  The    cerebrospinal    fluid    of    43    individuals, 
comprising  cases  of   alcoholic  psychoses,  dementia 
praecox,  epileptic  and  other  cerebral  affections,  was 
tested.      None  of   these  gave   a    positive  reaction 
with  Noguchi's  test;  two  gave  a  faint  reaction  with 
Wassermann's  test. 

(3)  Tests  made  on  the  serum  of  syphilitic  indi- 
viduals gave  the  following  results : 


Stage  of  Infection. 

Wassermann  Test. 

Noguchi  Test. 

Positive. 

Negative. 

Positive. 

Negative. 

Primary  syphilis: 
Untreated.                      

3 
o 

13 

i 
i 

i 
i 

i 
ii 

2 

4 

0 

14 

5 

i 

o 

I 

O 

7 

2 

Treated  f  

Secondary  syphilis: 
Untreated  and  little  treated  .  . 
Well   treated    or   still    under 
treatment   

Tertiary  syphilis 

(4)  The  serum  of  17  normal,  non-syphilitic  indi- 
viduals was  tested,  without  a  positive  reaction  with 
either  test.  It  may,  however,  be  stated  that  a 


168  APPENDIX. 

positive  reaction  was  obtained  in  an  advanced  case 
of  tuberculosis,  but  it  was  not  possible  to  say 
definitely  that  a  syphilitic  infection  was  not  also 
present. 

According  to  Noguchi,  the  Wassermann  test  occa- 
sionally gives  negative  results  in  general  paresis ;  the 
butyric  acid  test  always  gives  a  positive  reaction, 
giving  results  which  agree  perfectly  with  those 
obtained  by  cytodiagnosis,  and  with  the  clinical 
picture.  Noguchi's  test  for  spinal  fluid  can  be 
applied  even  to  old  specimens  (a  year  old)  with 
equally  good  results  as  in  fresh  fluid.  Wasser- 
mann 's  test  requires  fresh  spinal  fluid.  Whether 
this  simple  test  can  be  substituted  for  the  well- 
tried  Wassermann  reaction  can  only  be  determined 
by  a  large  number  of  careful  control  experiments. 


INDEX 


PAGE 

Abel,  deflection  of  complement 98 

Abrin 21 

Agglutination,  the  phenomenon 30 

purpose  of 33 

historical      33 

nature  of  reaction 36 

Agglutinins .  30 

specific,  group 40 

nature  of 35 

Agglutinoids 38 

Alexins 48,  52 

Amboceptor , .  61 

Anaphylactin 142 

Anaphylaxis .  142 

Anderson,  hypersusceptibility      141 

Antialbumoses 108 

Anticytotoxins 119 

Anticomplements 85 

Antigens 16 

Antihcemolysins      84 

Anti-immune-body ,    .    .  84 

Anti-isolysins 95 

Antiprecipitins 118 

Antitoxins i 

historical i 

concentration  of 145 

nature  of      17 

production  of 2 

relation  to  toxin 21 

testing  strength  of 5 

Antitoxic  globulins,  Gibson's 145 

Antivenins 135 

Aronson,  diphtheria  serum        5 

169 


1 70  INDEX 

PAGE 

Arrhenius,  toxin-antitoxin 28 

Atkinson,  antitoxic  globulins        12 

Autoanticomplements        88 

Autolysins 95 

Bacterial  precipitins 107 

Bactericides,  specific      49 

Bactericidal  sera,  value  of 104 

Bacteriotropic  substances 127 

Bacteriolysins 47 

historical 47 

Bail,  source  of  complements 92 

Beebe,  cytotoxic  sera 123 

Behring,  action  of  diphtheric  antiserum 6 

discovery  of  antitoxin i 

the  antitoxic  unit 22 

Belfanti  and  Carbone,  antitoxic  globulins 18 

haemotoxins 50 

Besredka,  nature  of  immune  body 81 

antihaemolysins       85 

Blood  test,  Deutsch's 102 

Neisser-Sachs       70 

precipitin 112 

Blood  transfusion,  dangers  of 50 

Bolduan,  value  of  opsonic  index 133 

Bordet,  nature  of  agglutination  reaction 37 

hcemolysis         31 

Pfeiffer's  phenomenon 49 

toxin-antitoxin  reaction 28 

Bordet-Gengou  phenomenon 68 

Bordet,  lactoserum 107 

Buchner,  alexins 48 

source  of  complements 9- 

Butyric  acid  test,  Noguchi's 164 

Buxton,  deflection  of  complement 102 

Calcar,  toxons 29 

Calmette,  antivenin 13? 

action  of  antitoxins 18 

Castellani,  absorption  test  for  group  agglutinins 41 


INDEX 

PAGB 

Clump  reaction .    .  30 

Collins,  specific  and  group  agglutinins 42 

Colloids,  relation  to  agglutination 37 

Complement 61 

deflection  of = 97 

multiplicity  of 67 

source  of 9^ 

structure  of 93 

Complementoid 93 

Concentration  of  antitoxin 145 

Copula      6 1 

Cytotoxin 119 

by  use  of  nucleo-proteid 123 

for  epithelium 122 

Death,  sudden .  138 

Deflection  of  complement 97 

Delezenne  and  Metchnikoff,  neurotoxin „    .    .  120 

Desmon 61 

Deutsch,  hagmolytic  blood  test 102 

Dialysis  of  toxons  and  toxins 29 

Dieudonne,  antitoxic  globulins .......  18 

Diphtheria  antitoxin,  production  of        2 

toxin,  production  of 2 

poison,  constitution  of 25 

Dungern,  v.,  haemolysis 51 

Durham,  discovery  of  agglutinins 34 

Ehrlich,  method  of  studying  toxins 23 

relation  of  toxins  to  antitoxin       29 

side-chain  theory,  applied  to  antitoxins 6 

ditto,  to  agglutinins        43 

ditto,  to  hasmolysins  and  bacteriolysins 65 

Ehrlich  and  Morgenroth  on  haemolysis        .    „ 56 

Electric  charge  of  toxins  and  antitoxins 29 

Field,  the  "  pro  zone  "  in  agglutination 39 

Field  and  Teague,  toxin-antitoxin 29 

Flexner  and  Noguchi,  snake  venoms       135 

Fluctuations  in  serum  constituents 90 


I?2  INDEX 

PAGE 

Friedberger,  salts  in  agglutination 37 

Fodor,  bactericidal  action 47 

Gay  and  Southard,  anaphylaxis 142 

Gengou-Bordet  phenomenon       68 

Gibson,  antitoxic  globulin 18,  14- 

Globulins,  antitoxic 18 

in  syphilitic  serum 16^ 

Group  agglutinins 39 

Gruber,  source  of  complements        92 

Gruber  and  Durham,  agglutination 34 

Gruber- Widal  reaction 34 

Grlinbaum,  significance  of  agglutination  test  in  typhoid       .  34 

Gscheidlen  and  Traube 47 

Haeckel,  phagocytosis 125 

Haemagglutinins 32 

Haemolysis «••.». 51 

Hsemolysin       .•.'«. 51 

Haemolytic  blood  test „    .  102 

Hoemotoxin „ 51 

Hasmorrhagin .  136 

Hahn,  sources  of  complement       92 

Haptins «, 16 

Haptophore  group  of  toxins 7 

Hektoen,  opsonins 128 

Horses,  for  diphtheria  antitoxin 3 

Hypersusceptibility    . 141 

Immune  body 6r 

nature  of 0 76 

partial „ 76 

where  produced 83 

Inter-body       74 

Isolysin 95 

Isoprecipitin 118 

Jackson,  cytotoxic  sera 124 

Johannessen 139 

Joos,  salts  in  agglutination  reaction 36 


INDEX  173 

PAGE 

Knorr,  on  antitoxins 5 

immunization  with  tetanus  toxin 2 

Kraus,  bacterial  precipitins      107 

Kyes,  snake  venoms 0 135 

Lactoserum 107 

Landois,  blood  transfusion 50 

Landsteiner,  hasmolysins 51 

spermatoxin 121 

source  of  complements » 93 

Leblanc,  nature  of  precipitins „...  no 

Leclainche  and  Vallee,  precipitins        ......    ....  108 

Ledingham,  antitoxic  globulins 19 

Leucocytes,  source  of  complements 92 

Leucotoxin      119 

Loffler  and  Abel,  deflection  of  complement 98 

Martin,  on  antitoxins 5 

Marx,  production  of  immune  body 83 

Metchnikoff,  cytotoxins , 119 

on  Pfeiffer's  test .  49 

phagocytosis „    . 125 

source  of  alexins .    . 92 

Mertens,  precipitins 108 

Moreschi-Gengou  phenomenon 69 

Morgenroth,  the  antitoxin  reaction     .    . 18 

on  hasmolysis 56 

Moxter,  alexins  and  leucocytes „  92 

spermatoxin  and  haemolysis 121 

Mliller,  structure  of  complements 93 

Multiplicity  of  complements „    .    .    .  67 

Myers,  precipitins ........  108 

Neisser-Sachs  Blood  test 70 

Neisser-Wechsberg  phenomenon 97 

Neisser-Wassermann  test  for  syphilis 69,  147 

Neufeld  and  Rimpau,  bacteriotropic  substances 127 

Neurotoxin 120 

Noguchi,  snake  venoms 135 

test  for  syphilis 164 

Nuttall,  precipitins 107 


174  INDEX 

PAGR 

Obermayer  and  Pick,  nature  of  precipitins no 

Opsonins 125 

distinct  antibodies 128 

historical „ 125 

structure  of 128 

Opsonic  index 128 

Park,  on  agglutinins 42 

diphtheria  antitoxin       2 

serum  rashes 139,  145 

antitoxic  globulins 12 

Pearce,  cytotoxic  sera 124 

Pfaundler,  group  agglutination 40 

thread  reaction 35 

Pfeiffer,  alexin  and  leucocyte 92 

Pfeiffer's  phenomenon 48 

Phytotoxins 21 

Pick,  fractionation  of  immune  sera 19 

v.  Pirquet  and  Schick,  serum  sickness <,..  138 

Poison  spectra,  Ehrlich's „ .  25 

Precipitins 106 

bacterial 107 

nature  of no 

test  tube  reaction  only      145 

Precipitins,  in  serum  sickness 145 

specificity  of 108 

Precipitin  blood  test      .    . 112 

Prototoxoids 36 

Pro  zone  in  agglutination 38 

Rashes  after  serum  injections  138 

Reactivation  of  sera 52 

Receptors 9 

various  orders  of 43 

Ricin 21 

Rosenau,  on  hypersusceptibility 141 

Rostoski,  bacterial  precipitins 107 

precipitin  reaction 145 

Sachs,  blood  test ,     .  70 

snake  venoms „ 135 


INDEX  J75 

PAGE 

Salts,  necessary  in  agglutination 37 

precipitin  test m 

Schattenfroh,  source  of  complements 92 

Schick,  serum  sickness 138 

Schiitze,  precipitins .    .    .    .  108 

Sera,  practical  value  of • 104 

Serum,  active  and  inactive 52 

Serum,  collection  of 4 

cytotoxic 119 

normal,  properties  of 47,  71 

normal  and  immune , 76 

Serum-sickness 138 

Side-chains,  functions  of 6 

Side  chain  theory,  antitoxins 6 

agglutinins  ...... 43 

bacteriolysins  and  hasmolysins 65 

Smith,  Theobald,  hypersusceptibility 141 

Snake  venoms     . 135 

Southard,  anaphylaxis       142 

Spectra,  of -toxins       25 

Spermatoxin 121 

Stimulins 126 

Substance  sensibilatrice 52 

Syntoxoids 27 

Syphilis,  test  for 69,  147 

Tchistowitch,  precipitins 106 

Teague,  toxin-antitoxin  reaction      „ 29 

Therapeutic  value  of  bactericidal  sera 104 

Thread  reaction      35 

Throne,  refined  antitoxin,  clinically    . 145 

Toxin,  according  to  Ehrlich 6 

nature  of  true ........  20 

relation  to  antitoxin 0    .    .  21 

production  of  diphtheria        2 

Toxoid,  according  to  Ehrlich «, 23 

affinity  for  antitoxin 24 

Toxon,  according  to  Ehrlich 23 

Toxophore  group  of  toxins 7 


176  INDEX 

PAGE 
Uhlenhuth,  precipitins  ....    ............      I0g 

blood  test     ....................      II2 


Van  Calcar,  toxons 
Von  Behring  (see  under  B). 
Von  Pirquet  (see  under  P). 
Venoms,  snake 


Wassermann,  antitoxin  reaction      ...........  18 

support  for  Ehrlich's  theory     ...........    „  I^ 

test  for  syphilis  ............    .....    69,  147 

Wassermann-  Uhlenhuth  blood  test     ..........  112 

Wechsberg,  deflection  of  complement     .........  98 

Weigert,  overproduction  theory  ............  9 

Wernicke,  on  antitoxins    .    .    0    ............  ^ 

Widal,  agglutination  reaction  .............  34 

Wright,  opsonins  ...................  126 

Zootoxins     ...................    „    .  21 

Ziilzer,  precipitins       .................  IO8 

Zymotoxic  group    ........    .   .......  ...  93 


OF  THE 

UNIVERSITY 


SHORT-TITLE     CATALOGUE 

OF  THE 

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8vo,  4  oo 

Matthews's  The  Textile  Fibres.   2d  Edition,  Rewritten 8vo,  4  oo 

Meyer's  Determination  of  Radicles  in  Carbon  Compounds.     (Tingle.).  .i2mo,  i  oo 

Miller's  Cyanide  Process I2mo,  i  oo 

Manual  of  Assaying I2mo,  i  oo 

Minet's  Production  of  Aluminum  and  its  Industrial  Use.     (Waldo.).  .  .  .  i2mo,  2  50 

Mixter's  Elementary  Text-book  of  Chemistry I2mo,  i  50 

Morgan's  Elements  of  Physical  Chemistry I2mo,  3  co 

Outline  of  the  Theory  of  Solutions  and  its  Results I2mo,  i  oo 

*  Physical  Chemistry  for  Electrical  Engineers I2mo,  i   50 

Morse's  Calculations  used  in  Cane-sugar  Factories i6mo,  mor.  i  50 

*  Muir's  History  of  Chemical  Theories  and  Laws 8vo,  4  oo 

Mulliksn's  General  Method  for  the  Identification  of  Pure  Organic  Compounds. 

Vol.  I Large  8vo,  5  oo 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo,  2  oo 

Ostwald's  Conversations  on  Chemistry.     Part  One.    (Ramsey.) i2mo,  i  50 

"             Part  Two.     (Turnbull.) i2mo,  200 

*  Palmer's  Practical  Test  Book  of  Chemistry i2mo,  i  oo 

*  Pauli's  Physical  Chemistry  in  the  Service  of  Medicine.     (Fischer.) .  .  .  .  i2mo,  i   25 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo,  paper,  50 
Tables   of  Minerals,  Including  the   Use   of  Minerals  and  Statistics  of 

Domestic  Production 8vo,  I  oo 

Pictet's  Alkaloids  and  their  Chemical  Constitution.     (Biddle.)  .  .. 8vo,  5  oo 

Poole's  Calorific  Power  of  Fuels 8vo,  3  oo 

Prescott  and  Winslow's  Elements  of  Water  Bacteriology,  with  Special  Refer- 
ence to  Sanitary  Water  Analysis i2mo,  i  50 

*  Reisig's  Guide  to  Piece-dyeing 8vo,  25  oo 

Richards  and  Woodman's  Air,  Water,  and  Food  from  a  Sanitary  Standpoint..8vo,  2  oo 

Ricketts  and  Miller's  Notes  on  Assaying 8vo,  3  oo 

Rideal's  Disinfection  and  the  Preservation  of  Food 8vo,  4  oo 

Sewage  and  the  Bacterial  Purification  of  Sewage 8vo,  4  oo 

Riggs's  Elementary  Manual  for  the  Chemical  Laboratory 8vo,  i  25 

Robine  and  Lenglen's  Cyanide  Industry.  (Le  Clerc.) 8vo,  4  oo 

Ruddiman's  Incompatibilities  in  Prescriptions 8vo,  2  oo 

Whys  in  Pharmacy J2mo,  i  oo 

5 


Ruer's  Elements  of  Metallography.     (Mathewson).     (In  Preparation.) 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish 8vo,    3  oo 

Salkowski's  Physiological  and  Pathological  Chemistry.     (Orndorff.) 8vo, 


Schimpf's  Essentials  of  Volumetric  Analysis i2mo, 

*       Qualitative  Chemical  Analysis 8vo, 

Text-book  of  Volumetric  Analysis i2mo, 

Smith's  Lecture  Notes  on  Chemistry  for  Dental  Students , .  .8vo, 


50 
25 
25 
50 
50 

Spencer's  Handbook  for  Cane  Sugar  Manufacturers i6mo,  mor.  3  oo 

Handbook  for  Chemists  of  Beet-sugar  Houses i6mo,  mor.  3  oo 

Stockbridge's  Rocks  and  Soils 8vo,    2  50 

*  Tillman's  Descriptive  General  Chemistry 8vo,    3  oo 

*  Elementary  Lessons  in  Heat 8vo,     i  50 

Treadwell's  Qualitative  Analysis.     (Hall.) 8vo,    3  oo 

Quantitative  Analysis.     (Hall.) 8vo,    4  oo 

Turneaure  and  Russell's  Public  Water-supplies 8vo,    5  oo 

Van  Deventer's  Physical  Chemistry  for  Beginners.     (Boltwood.) i2mo,    i  50 

Venable's  Methods  and  Devices  for  Bacterial  Treatment  of  Sewage 8vo,  3  oo 

Ward  and  Whipple's  Freshwater  Biology.     (In  Press.) 

Ware's  Beet-sugar  Manufacture  and  Refining.     Vol.  I Small  8vo,    4  oo 

Vol.  II SmallSvo,     500 

Washington's  Manual  of  the  Chemical  Analysis  of  Rocks.  , 8vo,    2  oo 

*  Weaver's  Military  Explosives 8vo,     3  oo 

Wells's  Laboratory  Guide  in  Qualitative  Chemical  Analysis 8vo,     i  50 

Short  Course  in  Inorganic  Qualitative  Chemical  Analysis  for  Engineering 

Students i2mo,     i  50 

Text-book  of  Chemical  Arithmetic i2mo,     i  25 

Whipple's  Microscopy  of  Drinking-water 8vo,    3  50 

Wilson's  Chlorination  Process I2mo      i  53 

Cyanide  Processes 12010      i  50 

Winton's  Microscopy  of  Vegetable  Foods 8vo      7  50 


CIVIL  ENGINEERING. 

BRIDGES  AND   ROOFS.     HYDRAULICS.     MATERIALS   OF    ENGINEER- 
ING.    RAILWAY   ENGINEERING. 

Baker's  Engineers'  Surveying  Instruments i2mo,  3  oo 

Bixby's  Graphical  Computing  Table Paper  19^X24!  inches.  25 

Breed  and  Hosmer's  Principles  and  Practice  of  Surveying 8vo,  3  oo 

*  Burr's  Ancient  and  Modern  Engineering  and  the  Isthmian  Canal 8vo,  3  50 


Comstock's  Field  Astronomy  for  Engineers 8vo, 

*  Corthell's  Allowable  Pressures  on  Deep  Foundations I2mo, 

Crandall's  Text-book  on  Geodesy  and  Least  Squares 8vo, 

Davis's  Elevation  and  Stadia  Tables 8vo, 

Elliott's  Engineering  for  Land  Drainage i2mo, 

Practical  Farm  Drainage I2mo, 

*Fiebeger's  Treatise  on  Civil  Engineering 8vo,    5  oo 

Flemer's  Phototopographic  Methods  and  Instruments 8vo,    5  oo 

Folwell's  Sewerage.      (Designing  and  Maintenance.) 8vo,    3  oo 

Freitag's  Architectural  Engineering 8vo,    3  50 

French  and  Ives's  Stereotomy 8vo,    2  50 

Goodhue's  Municipal  Improvements i2mo,     i  50 

Gore's  Elements  of  Geodesy •    8vo,    2  50 

*  Hauch  and  Rice's  Tables  of  Quantities  for  Preliminary  Estimates, I2mo,     i  25 

Hayford's  Text-book  of  Geodetic  Astronomy 8vo,    3  oo 

Bering's  Ready  Reference  Tables  (Conversion  Factors) i6mo,  mor.    2  50 

Howe's  Retaining  Walls  for  Earth i2mo,    i  25 

6 


*  Ives's  Adjustments  of  the  Engineer's  Transit  and  Level i6mo,  Bds.  25 

Ives  and  Hilts's  Problems  in  Surveying i6mo,  mor.  i  50 

Johnson's  (J.  B.)  Theory  and  Practice  of  Surveying Small  8vo,  4  oo 

Johnson's  (L.  J.)  Statics  by  Algebraic  and  Graphic  Methods 8vo,  2  oo 

Kinnicutt,  Winslow  and  Pratt's  Purification  of  Sewage.     (In  Preparation). 
Laplace's.  Philosophical    Essay    on    Probabilities.       (Truscott    and   Emory.) 

i2mo,  2  oo 

Mahan's  Descriptive  Geometry 8vo,  i  50 

Treatise  on  Civil  Engineering.     (1873.)     (Wood.) 8vo,  5  oo 

Merriman's  Elements  of  Precise  Surveying  and  Geodesy 8vo,  2  50 

Merriman  and  Brooks's  Handbook  for  Surveyors i6mo,  mor.  2  oo 

Morrison's  Elements  of  Highway  Engineering.       (In  Press.) 

Nugent's  Plane  Surveying 8vo,  3  50 

Ogden's  Sewer  Design I2mo,  2  oo 

Parsons's  Disposal  of  Municipal  Refuse 8vo,  2  oo 

Patton's  Treatise  on  Civil  Engineering 8vo,  half  leather,  7  50 

Reed's  Topographical  Drawing  and  Sketching 4to,  5  oo 

Rideal's  Sewage  and  the  Bacterial  Purification  of  Sewage 8vo,  4  oo 

Riemer's  Shaft-sinking  under  Difficult  Conditions.     (Corning  and  Peele.) .  .8vo,  3  oo 

Siebert  and  Biggin's  Modern  Stone-cutting  and  Masonry 8vo,  I  50 

Smith's  Manual  of  Topographical  Drawing.     (McMillan.) 8vo,  2  50 

Soper's  Air  and  Ventilation  of  Subways.     (In  Press.) 

Tracy's  Plane  Surveying I6mo,  mor.  3  oo 

*  Trautwine's  Civtt  Engineer's  Pocket-book i6mo,  mor.  5  oo 

Venable's  Garbage  Crematories  in  America 8vo,  2  oo 

Methods  and  Devices  for  Bacterial  Treatment  of  Sewage 8vo,  3  oo 

Wait's  Engineering  and  Architectural  Jurisprudence 8vo,  6  oo 

Sheep,  6  50 

Law  of  Contracts 8vo,  3  oo 

Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture  8vo,  5  oo 

Sheep,  5  50 

Warren's  Stereotomy — Problems  in  Stone-cutting 8vo,  2  50 

*  Waterbury's  Vest-Pocket  Hand-book   of   Mathematics   for   Engineers. 

3iX  si  inches,  mor.  i  oo 
Webb's  Problems  in  the  Use  and  Adjustment  of  Engineering  Instruments. 

i6mo,  mor.  i  25 

Wilson's  Topographic  Surveying 8vo,  3  50 

BRIDGES  AND  ROOFS. 

Boiler's  Practical  Treatise  on  the  Construction  of  Iron  Highway  Bridges.  .8vo,  2  oo 

Burr  and  Falk's  Design  and  Construction  of  Metallic  Bridges 8vo,  5  oo 

Influence  Lines  for  Bridge  and  Roof  Computations 8vo,  3  oo 

Du  Bois's  Mechanics  of  Engineering.     Vol.  II Sirall  4to,  10  oo 

Foster's  Treatise  on  Wooden  Trestle  Bridges 4to,  5  oo 

Fowler's  Ordinary  Foundations 8vp,  3  50 

French  and  Ives's  Stereotomy 8vo,  50 

Greene's  Arches  in  Wood,  Iron,  and  Stone 8vo,  50 

Bridge  Trusses 8vo,  50 

Roof  Trusses 8vo,  25 

Grimm's  Secondary  Stresses  in  Bridge  Trusses 8vo,  50 

Heller's  Stresses  in  Structures  and  the  Accompanyin    Deformations 8vo, 

Howe's  Design  of  Simple  Roof -trusses  in  Wood  and  Steel .8vo,  2  oo 

Symmetrical  Masonry  Arches 8vo,  2  50 

Treatise  on  Arches 8vo,  4  oo 

Johnson,  Bryan,  and  Turneaure's  Theory  and  Practice  in  the  Designing  of 

Modern  Framed  Structures Small  4to,  10  oo 

7 


Merriman  and  Jacoby's  Text-book  on  Roofs  and  Bridges: 

Part  I.      Stresses  in  Simple  Trusses 8vo,  2  50 

Part  II.    Graphic  Statics 8vo,  2  50 

Part  III.  Bridge  Design 8vo,  2  50 

Part  IV.   Higher  Structures 8vo,  2  50 

Morison's  Memphis  Bridge Oblong  4to,  10  oo 

Sondericker's  Graphic  Statics,  with  Applications  to  Trusses,  Beams,  and  Arches. 

8vo,  2  oo 

Waddell's  De  Pontibus,  Pocket-book  for  Bridge  Engineers i6mo,  mor,  2  oo 

*          Specifications  for  Steel  Bridges i2mo,  50 

Waddell  and  Harrington's  Bridge  Engineering.     (In  Preparation.) 

Wright's  Designing  of  Draw-spans.     Two  parts  in  one  volume 8vo,  3  50 


HYDRAULICS. 

Barnes's  Ice  Formation 8vo,  3  oo 

Bazin's  Experiments  upon  the  Contraction  of  the  Liquid  Vein  Issuing  from 

an  Orifice.     (Trautwine.) 8vo,  2  oo 

Bovey's  Treatise  on  Hydraulics 8vo,  5  oo 

Church's  Diagrams  of  Mean  Velocity  of  Water  in  Open  Channels. 

Oblong  4to,  paper,  i  50 

Hydraulic  Motors 8vo,  2  oo 

Mechanics  of  Engineering 8vo,  6  oo 

Coffin's  Graphical  Solution  of  Hydraulic  Problems i6mo,  morocco,  2  50 

Flather's  Dynamometers,  and  the  Measurement  of  Power i2mo,  3  oo 

Folwell's  Water-supply  Engineering 8vo,  4  oo 

FrizelPs  Water-power 8vo,  5  oo 

Fuertes's  Water  and  Public  Health i2mo,  i  50 

Water-filtration  Works i2mo,  2  50 

Ganguillet  and  Kutter's  General  Formula  for  the  Uniform  Flow  of  Water  in 

Rivers  and  Other  Channels.     ( Bering  and  Trautwine.) 8vo,  4  oo 

Hazen's  Clean  Water  and  How  to  Get  It Large  I2mo,  i  5o 

Filtration  of  Public  Water-supplies 8vo,  3  oo 

Hazlehurst's  Towers  and  Tanks  for  Water-works 8vo,  2  50 

Hefschel's  115  Experiments  on  the  Carrying  Capacity  of  Large,  Riveted,  Metal 

Conduits 8vo,  2  oo 

Hoyt  and  Grover's  River  Discharge 8vo,  2  oo 

Hubbard  and  Kiersted's  Water- works  Management  and  Maintenance 8vo,  4  uo 

*  Lyndon's  Development  and  Electrical  Distribution  of  Waler  Power.  .  .  .8vo,  3  oo 
Mason's  Water-supply.     (Considered  Principally  from  a  Sanitary  Standpoint.) 

8vo,  4  oo 

i,ierriman's  Treatise  on  Hydraulics 8vo,  5  oo 

*  Michie's  Elements  of  Analytical  Mechanics 8vo,  4  oo 

Molitor's  Hydraulics  of  Rivers,  Weirs  and  Sluices.     ^In  Press.) 

Schuyler's   Reservoirs  for  Irrigation,   Water-power,   and   Domestic   Water- 
supply Large  8vo,  5  oo 

*  Thoma^  and  Watt's  Improvement  of  Rivers 4to,  6  oo 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  oo 

Wegmann's  Design  and  Construction  of  Dams.     5th  Ed.,  enlarged 4to,  6  oo 

Water-supply  of  the  City  of  New  York  from  1658  to  1895 4to,  10  oo 

Whipple's  Value  of  Pure  Water Large  i2mo,  i  oo 

Williams  and  Hazen's  Hydraulic  Tables. 8vo,  i  50 

Wilson's  Irrigation  Engineering Small  8vo,  4  oo 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  oo 

Wood's  Elements  of  Analytical  Mechanics 8vo,  3  oo 

Turbines 8vo,  2  50 


MATERIALS  OF  ENGINEERING. 

Baker's  Roads  and  Pavements 8vo,  5  oo 

Treatise  on  Masonry  Construction 8vo,  5  oo 

Birkmire's  Architectural  Iron  and  Steel 8vo,  3  50 

Compound  Riveted  Girders  as  Applied  in  Buildings 8vo,  2  oo 

Black's  United  States  Public  Works Oblong  4to,  5  oo 

Bleininger's  Manufacture  of  Hydraulic  Cement.      (In  Preparation.) 

*  Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering 8vo,  7  50 

Byrne's  Highway  Construction 8vo,  5  oo 

Inspection  of  the  Materials  and  Workmanship  Employed  in  Construction. 

i6mo,  3  oo 

Church's  Mechanics  of  Engineering 8vo,  6  oo 

Du  Bois's  Mechanics  of  Engineering. 

Vol.   I.  Kinematics,  Statics,  Kinetics Small  4to,  7  50 

VoL  II.  The  Stresses  in  Framed  Structures,  Strength  of  Materials  and 

Theory  of  Flexures Small  4to,  10  oo 

*Eckel's  Cements,  Limes,  and  Plasters 8vo,  6  oo 

Stone  and  Clay  Products  used  in  Engineering.     (In  Preparation.) 

Fowler's  Ordinary  Foundations 8vo,  3  50 

Graves's  Forest  Mensuration 8vo,  4   oo 

Green's  Principles  of  American  Forestry 1 2mo,  i   *o 

*  Greene's  Structural  Mechanics 8vo,  2  50 

Holly  and  Ladd's  Analysis  of  Mixed  Paints,  Color  Pigments  and  Varnishes 

Large  i2mo,  2  50 

Johnson's  Materials  of  Construction Large  8vo,  6  oo 

Keep's  Cast  Iron 8vo,  2  50 

Kidder's  Architects  and  Builders'  Pocket-book i6mo,  5  oo 

Lanza's  Applied  Mechanics 8vo,  7  50 

Maire's  Modern  Pigments  and  their  Vehicles I2mo,  2  oo 

Martens's  Handbook  on  Testing  Materials.     (Henning.)     2  vols 8vo,  7  50 

Maurer's  Technical  Mechanics 8vo,  4  oo 

Merrill's  Stones  for  Building  and  Decoration 8vo,  5  oo 

Merriman's  Mechanics  of  Materials 8vo,  5  oo 

*  Strength  of  Materials I2mo,  i  oo 

Metcalf's  Steel.     A  Manual  for  Steel-users i2mo,  2  oo 

Patton's  Practical  Treatise  on  Foundations 8vo,  5  oo 

Rice's  Concrete  Block  Manufacture 8vo,  2  oo 

Richardson's  Modern  Asphalt  Pavements 8vo,  3  oo 

Richey's  Handbook  for  Superintendents  of  Construction i6mo,  mor.,  4  oo 

*  Ries's  Clays:  Their  Occurrence,  Properties,  and  Uses 8vo,  5  oo 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish 8vc,  3  oo 

*Schwarz's  Longleaf  Pine  in  Virgin  Forest, i2mo,  i   25 

Snow's  Principal  Species  of  Wood 8vo,  3  50 

Spalding's  Hydraulic  Cement i2mo,  2  oo 

Text-book  on  Roads  and  Pavements I2mo,  2  oo 

Taylor  and  Thompson's  Treatise  on  Concrete,  Plain  and  Reinforced 8vo,  5  oo 

Thurston's  Materials  of  Engineering.     In  Three  Parts 8vo,  8  oo 

Part  I.     Non-metallic  Materials  of  Engineering  and  Metallurgy.  .  .  .  .8vo,  2  oo 

Part  II.     Iron  and  Steel 8vo,  3  50 

Part  III.     A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Tillson's  Street  Pavements  and  Paving  Materials 8vo,  4  oo 

Turneaure  and  Maurer's  Principles  of  Reinforced  Concrete  Construction..  .8vo,  3  oo 
Wood's  (De  V.)  Treatise  on  the  Resistance  of  Materials,  and  an  Appendix  on 

the  Preservation  of  Timber 8vo,  2  oo 

Wood's  (M.  P.)  Rustless  Coatings:    Corrosion  and  Electrolysis  of  Iron  and 

SteeL 8vo,  4  oo 

9 


RAILWAY  ENGINEERING. 

Andrews's  Handbook  for  Street  Railway  Engineers 3x5  inches,  mor.  i  25 

Berg's  Buildings  and  Structures  of  American  Railroads 4to,  5  oo 

Brooks 's  Handbook  of  Street  Railroad  Location i6mo,  mor.  I  50 

Butt's  Civil  Engineer's  Field-book i6mo,  mor.  2  50 

CrandalTs  Railway  and  Other  Earthwork  Tables .  8vo,  i  so 

Transition  Curve i6mo,  mor.  i  50 

*  Crockett's  Methods  for  Earthwork  Computations 8vo,  i  50 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book i6mo,  mor.  5  oo 

Dredge's  History  of  the  Pennsylvania  Railroad:   (1879) Paper,  5  oo 

Fisher's  Table  of  Cubic  Yards Cardboard,  25 

Godwin's  Railroad  Engineers'  Field-book  and  Explorers'  Guide.  .  .  i6mo,  mor.  2  50 
Hudson's  Tables  for  Calculating  the  Cubic  Contents  of  Excavations  and  Em- 
bankments  8vo,  i  oo 

Ives   and  Hilts's   Problems   in  Surveying,  Railroad   Surveying  and   Geodesy 

i6mo,  mor.     i  50 

Molitor  and  Beard's  Manual  for  Resident  Engineers i6mo,     i  oo 

Nagle's  Field  Manual  for  Railroad  Engineers i6mo,  mor.     3  oo 

Philbrick's  Field  Manual  for  Engineers i6mo,  mor.    3  oo 

Raymond's  Railroad  Engineering.     3  volumes. 

Vol.      I.  Railroad  Field  Geometry.     (In  Preparation.) 

Vol.    II.  Elements  of  Railroad  Engineering 8vo,    3  50 

Vol  III.  Railroad  Engineer's  Field  Book.     (In  Preparation.) 

Searles's  Field  Engineering i6mo,  mor.     3  oo 

Railroad  Spiral i6mo,  mor.     i  50 

Taylor's  Prismoidal  Formulae  and  Earthwork 8vo,     i  50 

*Trautwine's  Field  Practice  of  Laying   Out  Circular  Curves   for  Railroads. 

i2mo.  mor,     2  50 

*  Method  of  Calculating  the  Cubic  Contents  of  Excavations  and  Embank- 

ments by  the  Aid  of  Diagrams 8vo,  2  oo 

Webb's  Economics  of  Railroad  Construction Large  i2mo,  2  50 

Railroad  Construction i6mo,  mor.  5  oo 

Wellington's  Economic  Theory  of  the  Location  of  Railways Small  8vo,  5  oo 

DRAWING. 

Barr's  Kinematics  of  Machinery 8vo,    2  50 

*  Bartlett's  Mechanical  Drawing 8vo,    3  oo 

*  "  "  "  Abridged  Ed 8vo,  i  50 

Coolidge's  Manual  of  Drawing 8vo,  paper,  i  oo 

Coolidge  and  Freeman's  Elements  of  General  Drafting  for  Mechanical  Engi- 
neers  Oblong  4to,  2  50 

Durley's  Kinematics  of  Machines 8vo,  4  oo 

Emch's  Introduction  to  Projective  Geometry  and  its  Applications 8vo,  2  50 

Hill's  Text-book  on  Shades  and  Shadows,  and  Perspective 8vo,  2  oo 

Jamison's  Advanced  Mechanical  Drawing 8vo,  2  oo 

Elements  of  Mechanical  Drawing 8vo,  2  50 

Jones's  Machine  Design: 

Part  I.     Kinematics  of  Machinery 8vo,  i  50 

Part  II.    Form,  Strength,  and  Proportions  of  Parts 8vo,  3  QO 

MacCord's  Elements  of  Descriptive  Geometry 8vo,  3  oc 

Kinematics ;  or,  Practical  Mechanism 8vo,  5  oo 

Mechanical  Drawing 4to,  4  oo 

Velocity  Diagrams 8vo,  i  50 

McLeod's  Descriptive  Geometry Large  i2mo,  i  50 

*  Mahan's  Descriptive  Geometry  and  Stone-cutting 8vo,  i  50 

Industrial  Drawing.     (Thompson.) 8vo,    3  50 

10 


Moyer's  Descriptive  Geometry 8vo,  2  oo> 

Reed's  Topographical  Drawing  and  Sketching 4to,  5  oo 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  oo 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design. 8vo,  3  oo 

Robinson's  Principles  of  Mechanism 8vo,  3  oo 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo,  3  oo 

Smith's  (R.  S.)  Manual  of  Topographical  Drawing.     (McMillan.) 8vo,  2  50 

Smith  (A.  W.)  and  Marx's  Machine  Design 8vo,  3  oo 

*  Titsworth's  Elements  of  Mechanical  Drawing Oblong  8vo,  i  25 

"Varren's  Drafting  Instruments  and  Operations 121110,  i  25. 

Elements  of  Descriptive  Geometry,  Shadows,  and  Perspective 8vo,  3  50 

Elements  of  Machine  Construction  and  Drawing 8vo,  7  50 

Elements  of  Plane  and  Solid  Free-hand  Geometrical  Drawing.  .  .    i.2mo,  i  oo 

General  Problems  of  Shades  and  Shadows 8vo,  3  oo 

Manual  of  Elementary  Problems  in  the  Linear  Perspective  of  Form  and 

Shadow 121110,  i  oo 

Manual  of  Elementary  Projection  Drawing i2mo,  i  so- 
Plane  Problems  in  Elementary  Geometry i2tno,  i  25 

Problems,  Theorems,  and  Examples  in  Descriptive  Geometry 8vo,  2  50 

Weisbach's    Kinematics    and    Power    of    Transmission.        (Hermann    and 

Klein.) 8vo,  5  oo 

Wilson's  (H.  M.)  Topographic  Surveying 8vo,  3  50 

Wilson's  (V.  T.)  Free-hand  Lettering 8vo,  i  oo 

Free-hand  Perspective 8vo,  2  50 

Woolf's  Elementary  Course  in  Descriptive  Geometry Large  8vo,  3  oo 

ELECTRICITY  AND  PHYSICS. 

*  Abegg's  Theory  of  Electrolytic  Dissociation,     (von  Ende.) i2mo,  i   25 

Andrews's  Hand-Book  for  Street  Railway  Engineering 3X5  inches,  mor.,  i  25 

Anthony  and  Brackett's  Text-book  of  Physics.     (Magie.) Large  i2mo,  3  oo 

Anthony's  Lecture-notes  on  the  Theory  of  Electrical  Measurements.  .  .  .i2mo,  i  oo 

Benjamin's  History  of  Electricity 8vo,  3  oo 

Voltaic  Cell 8vo,  3  oo 

Betts's  Lead  Refining  and  Electrolysis 8vo,  4  oo 

Classen's  Quantitative  Chemical  Analysis  by  Electrolysis.     (Boltwood.).8vo,  3  oo 

*  Collins's  Manual  of  Wireless  Telegraphy i2mo,  i  50 

Mor.  2  oo 

Crehore  and  Squier's  Polarizing  Photo-chronograph 8vo,  3  oo 

*  Danneel's  Electrochemistry.     (Merriam.) I2mo,  i  25 

Dawson's  "Engineering"  and  Electric  Traction  Pockex-book i6mo,  mor  5  oo 

Dolezalek's  Theory  of  the  Lead  Accumulator  (Storage  Battery),    (von  Ende.) 

i2mo,  2  50 

Duhem's  Thermodynamics  and  Chemistry.     (Burgess.) 8vo,  4  oo 

Flather's  Dynamometers,  and  the  Measurement  of  Power i2mo,  3  oo 

Gilbert's  De  Magnete.     (Mottelay.) 8vo,  2  50 

*  Hanchett's  Alternating  Currents i2mo,  r  oo 

Bering's  Ready  Reference  Tables  (Conversion  Factors) i6mo,  mor.  2  50 

Hobart  and  Ellis's  High-speed  Dynamo  Electric  Machinery.     (In  Press.) 

Holman's  Precision  of  Measurements 8vo,  2  oo 

Telescopic   Mirror-scale  Method,  Adjustments,  and  Tests. .  .'.Large  8vo,  75 

*  Karapetoff's  Experimental  Electrical  Engineering 8vo,  6  oo 

Kinzbrunner's  Testing  of  Continuous-current  Machines 8vo,  2  oo 

Landauer's  Spectrum  Analysis.     (Tingle.) 8vo,  3  oo 

Le  Chatelier's  High-temperature  Measurements.  (Boudouard — Burgess.)  i2mo,  3  oo 

Lob's  Electrochemistry  of  Organic  Compounds.     (Lorenz.) 8vo,  3  oo 

*  London's  Development  and  Electrical  Distribntion  of  Water  Power  .  .  .  .8vo,  3  oo 

*  Lyons'?  Treatise  on  Electromagnetic  Phenomena.   Vols.  I.  and  II.  8vo,  each,  6  oo 

*  Michie's  Elements  of  Wave  Motion  Relating  to  Sound  and  Light 8vo,  4  oo 

11 


Morgan's  Outline  of  the  Theory  of  Solution  and  its  Results i2mo,  i  oo 

*  Physical  Chemistry  for  Electrical  Engineers i2mo,  i  50 

Niaudet's  Elementary  Treatise  on  Electric  Batteries.     (Fishback).  .  .  .  i2mo.  2  50 

*  Morris's  Introduction  to  the  Study  of  Electrical  Engineering Svo,  2  50 

*  Parshall  and  Hobart's  Electric  Machine  Design 4to,  half  morocco,  12  50 

Reagan's  Locomotives:    Simple,  Compound,  and  Electric.      New  Edition. 

Large  12 mo,  3  50 

*  Rosenberg's  Electrical  Engineering.     (Haldane  Gee— Kinzbrunner.).  .  .8vo,  2  oo 

Ryan,  Norris,  and  Hoxie's  Electrical  Machinery.     Vol.  1 8vo,  2  50 

Swapper's  Laboratory  Guide  for  Students  in  Physical  Chemistry i2mo,  i  oo 

Thurston's  Stationary  Steam-engines 8vo,  2  50 

*  Tillman's  Elementary  Lessons  in  Heat 8vo,  i  50 

Tory  and  Pitcher's  Manual  of  Laboratory  Physics Large  I2mo,  2  oo 

Ulke's  Modern  Electrolytic  Copper  Refining Svo,  3  oo 

LAW. 

*  Davis's  Elements  of  Law Svo,    2  50 

*  Treatise  on  the  Military  Law  of  United  States 8vo,    7  oo 

*  Sheep,     7  50 

*  Dudley's  Military  Law  and  the  Procedure  of  Courts-martial  .  .  .  .Large  i2mo,     2  50 

Manual  for  Courts-martial ibmo,  mor.     i  50 

Wait's  Engineering  and  Architectural  Jurisprudence < Svo,    6  oo 

Sheep,    6  50 

Law  of  Contracts Svo,    3  oo 

Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture  Svo      5  oo 

Sheep,    5  50 
MATHEMATICS. 

Baker's  Elliptic  Functions Svo, 

Briggs's  Elements  of  Plane  Analytic  Geometry.    (Bocher) i2mo, 

*  Buchanan's  Plane  and  Spherical  Trigonometry 8vo, 

Byerley's  Harmonic  Functions Svo, 

Chandler's  Elements  of  the  Infinitesimal  Calculus 12 mo, 

Compton's  Manual  of  Logarithmic  Computations 1 2mo, 

Davis's  Introduction  to  the  Logic  of  Algebra Svo, 

*  Dickson's  College  Algebra Large  i2mo, 

*  Introduction  to  the  Theory  of  Algebraic  Equations Large  i2mo, 

Emch's  Introduction  to  Projective  Geometry  and  its  Applications Svo, 

Fiske's  Functions  of  a  Complex  Variable Svo, 

Halsted's  Elementary  Synthetic  Geometry Svo, 

Elements  of  Geometry Svo, 

*  Rational  Geometry I2mo, 

Hyde's  Grassmann's  Space  Analysis Svo, 

*  Jonnson's  (,/   B,)  Three-place  Logarithmic  Tables:  Vest-pocket  size,  paper,         15 

100  copies,     5  oo 

*  Mounted  on  heavy  cardboard,  8  X 10  inches,         25 

10  copies,  2  oo 
Johnson's  (W.  W.)  Abridged  Editions  of  Differential  and  Integral  Calculus 

Large  i2mo,  i  vol.  2  50 

Curve  Tracing  in  Cartesian  Co-ordinates i2mo,  i  oo 

Differential  Equations Svo,  i  oo 

Elementary  Treatise  on  Differential  Calculus.     (In  Press.) 

tleB  icntary  Treatise  on  the  Integral  Calculus Large  i2mo>  i  50 

*  Theoretical  Mechanics i2mo,  3  oo 

Theory  of  Errors  and  the  Method  of  Least  Squares i2mo,  i  50 

Treatise  on  Differential  Calculus Large  12010,  3  oo 

Treatise  on  the  Integral  Calculus Large  i2mo,  3  oo 

Treatise  on  Ordinary  and  Partial  Differential  Equations. .  Large  i2mo,  3  50 

12 


£aplace's  Philosophical  Essay  on  Probabilities.     (Truscott  and  Emory.). i2mo,     2  oo 

*  Ludlow  and  Bass's  Elements  of  Trigonometry  and  Logarithmic  and  Other 

Tables 8vo,     3  oo 

Trigonometry  and  Tables  published  separately Each,     2  oo 

*  Ludlow's  Logarithmic  and  Trigonometric  Tables 8vo,     i  oo 

Macfarlane's  Vector  Analysis  and  Quaternions 8vo,     i  oo 

McMahon's  Hyperbolic  Functions 8vo,     i  oo 

Manning's  IrrationalNumbers  and  their  Representation  bySequences  and  Series 

i2mo,      i   25 
Mathematical  Monographs.     Edited  by  Mansfield  Merriman  and  Robert 

S.  Woodward Octavo,  each     i  oo 

Wo.  i.  History  of  Modern  Mathematics,  by  David  Eugene  Smith. 
No.  2.  Synthetic  Projective  Geometry,  by  George  Bruce  Halsted. 
No.  3.  Determinants,  by  Laenas  Gifford  Weld.  No.  4.  Hyper- 
bolic Functions,  by  James  McMahon.  Ko.  5.  Harmonic  Func- 
tions, by  William  E.  Byerly.  No.  6.  Grassmann's  Space  Analysis, 
by  Edward  W.  Hyde.  No.  7.  Probability  and  Theory  of  Errors, 
by  Robert  S.  Woodward.  No.  8.  Vector  Analysis  and  Quaternions, 
by  Alexander  Macfarlane.  No.  9.  Differential  Equations,  by 
William  Woolsey  Johnson.  No.  10.  The  Solution  of  Equations, 
by  Mansfield  Merriman.  No.  n.  Functions  of  a  Complex  Variable, 
by  Thomas  S.  Fiskc. 

Maurer's  Technical  Mechanics 8vo,    4  oo 

Meti (man's  Method  of  Least  Squares 8vo,    2  oo 

Solution  of  Equations 8vo,    i  oo 

Rice  and  Johnson's  Differential  and  Integral  Calculus.     2  vols.  in  one. 

Large  i2mo,     i  50 

Elementary  Treatise  on  the  Differential  Calculus. Large  I2mo,     3  oo 

Smith's  History  of  Modern  Mathematics 8vo,     i  oo 

*  Veblen  and  Lennes's  Introduction  to  the  Real  Infinitesimal  Analysis  of  One 

Variable 8vo,    2  oo 

*  Waterbury's  Vest  Pocket  Hand-Book  of  Mathematics  for  Engineers. 

2^X5$  inches,  mor.,     i  oo 

Weld's  Determinations 8vo,  .  i  oo 

\Vood's  Elements  of  Co-ordinate  Geometry 8vo,    2  oo 

Woodward's  Probability  and  Theory  of  Errors 8vo,    i  oo 

MECHANICAL  ENGINEERING. 

MATERIALS  OF  ENGINEERING,  STEAM-ENGINES  AND  BOILERS. 

Bacon's  Forge  Practice i2mo,  i  50 

Baldwin's  Steam  Heating  for  Buildings i2mo,  2  50 

Bair's  Kinematics  of  Machinery 8vo,  2  50 

*  Bartlett's  Mechanical  Drawing 8vo,  3  oo 

*  "  "  "        Abridged  Ed 8vo,    i  50 

Benjamin's  Wrinkles  and  Recipes i2mo,    2  oo 

*  Burr's  Ancient  and  Modern  Engineering  and  the  Isthmian  Canal 8vo,    3  50 

Carpenter's  Experimental  Engineering 8vo,    6  oo 

Heating  and  Ventilating  Buildings 8vo,  4  oo 

Clerk's  Gas  and  Oil  Engine Large  i2mo,  4  oo 

Compton's  First  Lessons  in  Metal  Working I2mo,  i  50 

Compton  and  De  Groodt's  Speed  Lathe I2mo,  i  50 

Coolidge's  Manual  of  Drawing 8vo,  paper,  i  oo 

Ccolidge  and  Freeman's  Elements  of  General  Drafting  for  Mechanical  En- 
gineers  Oblong  4to,  2  50 

Cromwell's  Treatise  on  Belts  and  Pulleys i2mo,  i  50 

Treatise  on  Toothed  Gearing 12010,  i  50 

Durley's  Kinematics  of  Machines 8vo,  4  oo 

13 


Flather's  Dynamometers  and  the  Measurement  of  Power, i2mo,  3  oa 

Rope  Driving i2mo,  2  oa 

Gill's  Gas  and  Fuel  Analysis  for  Engineers i2mo,  i  25 

Goss'n  Locomotive  Sparks 8vo,  2  oo> 

Hall's  Car  Lubrication i2mo,  i  oo 

Bering's  Ready  Reference  Tables  (Conversion  Factors) i6mo,  mor.,  2  50 

Hobart  and  Eltis's  High  Speed  Dynamo  Electric  Machinery.      (In  Press.) 

Button's  Gas  Engine 8vo,  5  oo 

Jamison's  Advanced  Mechanical  Drawing 8vo,  2  oo 

Elements  of  Mechanical  Drawing 8vo,  2  50 

Jones's  Machine  Design: 

Part  I.     Kinematics  of  Machinery 8vo,  i  50 

Part  II.     Form,  Strength,  and  Proportions  of  Parts 8vo,  3  oo 

Kent's  Mechanical  Engineers'  Pocket-book i6mo,  mor  ,  5  oo 

Kerr's  Power  and  Power  Transmission 8vo,  2  oo 

Leonard's  Machine  Shop  Tools  and  Methods   8vo,  4  oo 

*  Lorenz's  Modern  Refrigerating  Machinery.    (Pope,  Haven,  and  Dean.)  .  .8vo,  4  oo 
MacCord's  Kinematics;   or,  Practical  Mechanism 8vo,  5  oo 

Mechanical  Drawing 4to,  4  oo 

Velocity  Diagrams 8vo,  i  50 

MacFar land's  Standard  Reduction  Factors  for  Gases 8vo,  i  50 

Mahan's  Industrial  Drawing.     (Thompson.) 8vo,  3  50 

*  Parshall  and  Hobart's  Electric  Machine  Design Small  4to,  half  leather,  12  50 

Peele's  Compressed  Air  Plant  for  Mines.     (In  Press.) 

Poole's  Calorific  Power  of  Fuels 8vo,  3  oo 

*  Porter's  Engineering  Reminiscences,  1855  to  1882 8vo,  3  oo 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  oo- 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design. 8vo,  3  oo 

Richard's  Compressed  Air i2mo,  i  50 

Robinson's  Principles  of  Mechanism 8vo,  3  oo 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo,  3  oo 

Smith's  (O.)  Press- working  of  Metals 8vo,  3  oo 

Smith  (A.  W.)  and  Marx's  Machine  Design 8vo,  3  oo 

Sorel '.  s  Carbureting  and  Combustion  in  Alcohol  Engines .    ( Woodward  and  Preston) . 

Large  12 mo,  3  oo 

Thurston's  Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics. 

i2mo,  i  oo 

Treatise  on  Friction  and  Lost  Work  in  Machinery  and  Mill  Work...  8vo,  3  oo 

Tillson's  Complete  Automobile  Instructor i6mo,  i  50 

mor.,  2  oo 

*  Titsworth's  Elements  of  Mechanical  Drawing Oblong  8vo,  i    25 

Warren's  Elements  of  Machine  Construction  and  Drawing 8vo,  7  5» 

*  Waterbury's  Vest  Pocket  Hand  Book  of  Mathematics  for  Engineers. 

2^X  5s  inches,  mor.,  i   oo 
Weisbach's    Kinematics    and    the    Power    of    Transmission.     (Herrmann — 

Klein.) 8vo,  5  oo 

Machinery  of  Transmission  and  Governors.     (Herrmann — Klein.).  .8vo,  5  oo 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  oo 

Wood's  Turbines 8vo,  2  50 

MATERIALS   OF   ENGINEERING. 

*  Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering 8vo,  7  50 

Church's  Mechanics  of  Engineering 8vo,  6  oo 

*  Greene's  Structural  Mechanics 8vo,  2  50 

Holley  and  Ladd's  Analysis  of  Mixed  Paints,  Color  Pigments,  and  Varnishes. 

Large  tamo,  2  50 

Johnson's  Materials  of  Construction 8vo,  6  oo 

Keep's  Cast  Iron '.  .  8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

14 


Maire's  Modern  Pigments  and  their  Vehicles i2mo,  2  oo 

Martens's  Handbook  on  Testing  Materials.     (Henning.) 8vo,  7  50 

Maurer's  Technical  Mechanics 8vo,  4  oo 

Merriman's  Mechanics  of  Materials 8vo,  5  oo 

*         Strength  of  Materials I2mo,  i  oo 

Metcalf's  Steel.     A  Manual  for  Steel-users I2mo,  2  oo 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish 8vo,  3  oo 

Smith's  Materials  of  Machines I2mo,  i  oo 

Thurston's  Materials  of  Engineering 3  vols.,  8vo,  8  oo 

Part  I.     Non-metallic  Materials  of  Engineering,  see  Civil  Engineering, 
page  9. 

Part  II.     Iron  and  Steel 8vo,  3  50 

Part  III.     A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Wood's  (De  V.)  Elements  of  Analytical  Mechanics 8vo,  3  oo 

Treatise  on   the    Resistance    of    Materi-ls  and    an  Appendix  on  the 

Preservation  of  Timber 8vo,  2  oo 

Wood's  (M.  P.)  Rustless  Coatings:    Corrosion  and  Electrolysip  of  Iron  and 

Steel 8vo,  4  oo 

STEAM-ENGINES  AND  BOILERS. 

Berry's  Temperature-entropy  Diagram i2mo,  i  25 

Carnot's  Reflections  on  the  Motive  Power  of  Heat     (Thurston.) i2mo,  i  50 

Chase's  Art  of  Pattern  Making I2mo,  2  50 

Creighton's  Steam-engine  and  other  Heat-motors 8vo,  500 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.  . .  .i6mo,  mor.,  5  oo 

Ford's  Boiler  Making  for  Boiler  Makers i8mo,  i  oo 

Goss's  Locomotive  Performance 8vo,  5  oo 

Hemenway's  Indicator  Practice  and  Steam-engine  Economy i2mo,  2  oo 

Button's  Heat  and  Heat-engines 8vo.  5  oo 

Mechanical  Engineering  of  Power  Plants 8vo,  5  oo 

Kent's  Steam  boiler  Economy 8vo,  4  oo 

Kneass's  Practice  and  Theory  of  the  Injector 8vo,  i  50 

MacCord's  Slide-valves 8vo,  2  oo 

Meyer's  Modern  Locomotive  Construction 4to,  10  oo 

Moyer's  Steam  Turbines.     (Tn  Press.) 

Peabody's  Manual  of  the  Steam-engine  Indicator I2mo.  i  50 

Tables  of  the  Properties  of  Saturated  Steam  and  Other  Vapors 8vo,  i  oo 

Thermodynamics  of  the  Steam-engine  and  Other  Heat-engines 8vo,  5  oo 

Valve-gears  for  Steam-engines 8vo,  2  50 

Peabody  and  Miller's  Steam-boilers 8vo,  4  oo 

Pray's  Twenty  Years  with  the  Indicator Large  8vo,  2  50 

Pupin's  Thermodynamics  of  Reversible  Cycles  in  Gases  and  Saturated  Vapors. 

(Osterberg.) I2mo,  i  25 

Reagan's  Locomotives:    Simple,  Compound,  and  Electric.     New  Edition. 

Large  12 mo,  3  50 

Sinclair's  Locomotive  Engine  Running  and  Management I2mo,  2  oo 

Smart's  Handbook  of  Engineering  Laboratory  Practice I2mo,  2  50 

Snow's  Steam-boiler  Practice 8vo,  3  oo 

Spangler's  Notes  on  Thermodynamics I2mo,  i  oo 

Valve-gears 8vo,  2  50 

Spaagler,  Greene,  and  Marshall's  Elements  of  Steam-engineering 8vo,  3  oo 

Thomas's  Steam-turbines 8vo,  4  oo 

Thurston's  Handbook  of  Engine  and  Boiler  Trials,  and  the  Use  of  the  Indi- 
cator and  the  Prony  Brake 8vo,  5  oo 

Handy  Tables 8vo,  i  50 

Manual  of  Steam-boilers,  their  Designs,  Construction,  and  Operation..8vo,  5  oo 

15 


Thurston's  Manual  of  the  Steam-engine 2  vols.,  8vo,  10  oo 

Part  I.     History,  Structure,  and  Theory 8vo,  6  oo 

Part  II.     Design,  Construction,  and  Operation .8vo,  6  oo 

Stationary  Steam-engines 8vo,  2  50 

Steam-boiler  Explosions  in  Theory  and  in  Practice 12mo,  i  so 

Wehrenfenning's  Analysis  and  Softening  of  Boiler  Feed-water  (Patterson)   8vo,  4  oo 

Weisbach's  Heat,  Steam,  and  Steam-engines.     (Du  Bois.) 8vo,  5  oo 

Whitham's  Steam-engine  Design 8vo,  5  oo 

Wood's  Thermodynamics,  Heat  Motors,  and  Refrigerating  Machines.  ..8vo,  4  oo 

MECHANICS  PURE  AND  APPLIED. 

Church's  Mechanics  of  Engineering 8vo,  6  oo 

Notes  and  Examples  in  Mechanics 8vo,  2  oo 

Dana's  Text-book  of  Elementary  Mechanics  for  Colleges  and  Schools.  .i2mo,  i  50 
Du  Bois's  Elementary  Principles  of  Mechanics : 

Vol.      I.     Kinematics 8vo,  3  50 

Vol.    II.     Statics 8vo,  4  oo 

Mechanics  of  Engineering.     Vol.    I Small  4to,  7  50 

Vol.  II Small  4to,  10  oo 

*  Greene's  Structural  Mechanics 8vo,  2  50 

James's  Kinematics  of  a  Point  and  the  Rational  Mechanics  of  a  Particle. 

Large  12mo,  2  oo 

*  Johnson's  (W.  W.)  Theoretical  Mechanics 12mo,  3  oo 

Lanza's  Applied  Mechanics : 8vo,  7  50 

*  Martin's  Text  Book  on  Mechanics,  Vol.  I,  Statics 12mo,  i   25 

*  Vol.  2,  Kinematics  and  Kinetics  .  .i2mo,  1  50 
Maurer's  Technical  Mechanics 8vo,  4  oo 

*  Merriman's  Elements  of  Mechanics 12mo,  i  oo 

Mechanics  of  Materials 8vo,  5  oo 

*  Michie's  Elements  of  Analytical  Mechanics 8vo,  4  oo 

Robinson's  Principles  of  Mechanism 8vo,  3  oo 

Sanborn's  Mechanics  Problems Large  12mo,  i  50 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo,  3  oo 

Wood's  Elements  of  Analytical  Mechanics 8vo,  3  oo 

Principles  of  Elementary  Mechanics 12mo,  i  25 

MEDICAL. 

Abderhalden's  Physiological  Chemistry  in  Thirty  Lectures.     (Hall  and  Defren). 

(in  Press). 
von  Behring's  Suppression  of  Tuberculosis.     (Bolduan.) I2mo,     i  oo 

*  Bolduan's  Immune  Sera i2mo,     i  50 

Davenport's  Statistical  Methods  with  Special  Reference  to  Biological  Varia- 
tions   i6mo,  mor.,     i  50 

Ehrlich's  Collected  Studies  on  Immunity.     (Bolduan.) 8vo,  6  oo 

*  Fischer's  Physiology  of  Alimentation Large  i2mo,  cloth,  2  oo 

de  Fursac's  Manual  of  Psychiatry.     (Rosanoff  and  Collins.) Large  i2mo,  2  50 

Hammarsten's  Text-book  on  Physiological  Chemistry.     (Mandel.) 8vo,  4  oo 

Jackson's  Directions  for  Laboratory  Work  in  Physiological  Chemistry.  ..8vo,  i  25 

Lassar-Cohn's  Practical  Urinary  Analysis.     (Lorenz.) I2mo,  i  oo 

Mandel's  Hand  Book  for  the  Bio-Chemical  Laboratory i2mo,  i  50 

*  Pauli's  Physical  Chemistry  in  the  Service  of  Medicine.     (Fischer.) .  .  .  .  xarno,  i  25 

*  Pozzi-Escot's  Toxins  and  Venoms  and  their  Antibodies.     (Cohn.) i2mo,  i  oo 

Rostoski's  Serum  Diagnosis.     (Bolduan.) I2mo,  i  oo 

Ruddiman's  Incompatibilities  in  Prescriptions , 8vo,  2  oo 

Whys  in  Pharmacy I2mo,  i  oo 

Salkowski's  Physiological  and  Pathological  Chemistry.     (Orndorff.) 8vo,  2  50 

*  Satterlee's  Outlines  of  Human  Embryology 12 mo,  i  25 

Smith's  Lecture  Notes  on  Chemistry  for  Dental  Students 8vo,  2  50 

16 


Steel's  Treatise  on  the  Diseases  of  the  Dog 8vo,  3  50 

*  Whipple's  Typhoid  Fever ; Large  i2mo,  3  oo 

Woodhull's  Notes  on  Military  Hygiene i6mo,  i  50 

*  Personal  Hygiene i2mo,  i  oo 

Worcester  and  Atkinson's  Small  Hospitals  Establishment  and  Maintenance, 

and  Suggestions  for  Hospital  Architecture,  with  Plans  for  a  Small 

Hospital izmo,  i  25 

METALLURGY. 

Betts's  Lead  Refining  by  Electrolysis 8vo.  4  oo 

Holland's  Encyclopedia  of  Founding  and  Dictionary  of  Foundry  Terms    Used 

in  the  Practice  of  Moulding I2mo,  3  oo 

Iron  Founder 12mo.  2  50 

Supplement I2mo,  2  50 

Douglas's  Untechnical  Addresses  on  Technical  Subjects I2mo,  i  oo 

Goesel's  Minerals  and  Metals:     A  Reference  Book i6mo,  mor.  3  oo 

*  Iles's  Lead-smelting 12mo,  2  50 

Keep's  Cast  Iron 8vo,  2  50 

Le  Chatelier's  High-temperature  Measurements.  (Boudouard — Burgess.)  12mo,  3  oo 

Metcalf's  Steel.     A  Manual  for  Steel-users 12mo,  2  oo 

Miller's  Cyanide  Process 12mo  i  oo 

Minet's  Production  of  Aluminum  and  its  Industrial  Use.     (Waldo.)...  .  12mo,  2  50 

Robine  and  Lenglen's  Cyanide  Industry.     (Le  Clerc.) 8vo,  4  oo 

Ruer's  Elements  of  Metallography.     (Mathewson).     (In  Press.) 

Smith's  Materials  of  Machines 12mo,  i  oo 

Thurston's  Materials  of  Engineering.     In  Three  Parts 8vo,  8  oo 

part  I.     Non-metallic  Materials  of  Engineering,  see  Civil  Engineering, 
page  9. 

Part    II.     Iron  and  Steel 8vo,  3  50 

Part  III.     A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  ineir 

Constituents 8vo,  2  50 

Ulke's  Modern  Electrolytic  Copper  Refining 8vo,  3  oo 

West's  American  Foundry  Practice I2mo,  2  50 

Moulders  Text  Book I2mo,  2  50 

Wilson's  Chlorination  Process. 12mo,  i  50 

Cyanide  Processes I2mo,  i  50 

MINERALOGY. 

Barringer's  Description  of  Minerals  of  Commercial  Value.    Oblong,  morocco,  2  50 

Boyd's  Resources  of  Southwest  Virginia 8vo  3  oo 

Boyd's  Map  of  Southwest  Virginia Pocket-book  form.  2  oo 

*  Browning's  Introduction  to  the  Rarer  Elements 8vo,    i  50 

Brush's  Manual  of  Determinative  Mineralogy.     (Penfield.) 8vo,  4  oo 

Butler's  Pocket  Hand-Book  of  Minerals 16mo,  mor.  3  oo 

Chester's  Catalogue  of  Minerals 8vo,  paper,  i  oo 

Cloth,  i  25 

Crane's  Gold  and  Silver.     (In  Press.) 

Dana's  First  Appendix  to  Dana's  New  "  System  of  Mineralogy. ." .  .  Large  8vo,  i  oo 

Manual  of  Mineralogy  and  Petrography I2mo  2  oo 

Minerals  and  How  to  Study  Them I2mo,  i  50 

System  of  Mineralogy Large  8vo,  half  leather,  12  50 

Text-book  of  Mineralogy 8vo,  4  oo 

Douglas's  Untechnical  Addresses  on  Technical  Subjects 12010,  i  oo 

Eakle's  Mineral  Tables 8vo,  i  25 

Stone  and  Clay  Products  Used  in  Engineering.     (In  Preparation). 

Egleston's  Catalogue  of  Minerals  and  Synonyms 8vo,  2  50 

Goesel's  Minerals  and  Metals :     A  Reference  Book i6mo,  mor.  3  oo 

Groth's  Introduction  to  Chemical  Crystallography  (Marshall) I2mo,  i  25 

17 


*  Iddings's  Rock  Minerals 8vo,  5  oo 

Johannsen's  Determination  of  Rock-forming  Minerals  in  Thin  Sections 8vo,  4  oo 

*  Martin's  Laboratory  Guide  to  Qualitative  Analysis  with  the  Blowpipe.  12010,  60 
Merrill's  Non-metallic  Minerals:   Their  Occurrence  and  Uses 8vo,  4  oo 

Stones  for  Building  and  Decoration 8vo,  500 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo,  paper,  50 
Tables    of    Minerals,    Including   the  Use  of  Minerals  and  Statistics  of 

Domestic  Production 8vo,  i  oo 

Pirsson's  Rocks  and  Rock  Minerals.     (In  Press.) 

*  Richards's  Synopsis  of  Mineral  Characters i2mo,  mor,  i  25 

*  Ries's  Clays:  Their  Occurrence,  Properties,  and  Uses 8vo,  5  oo 

*  Tillman's  Text-book  of  Important  Minerals  and  Rocks 8vo,  2  oo 

MINING. 

*  Beard's  Mine  Gases  and  Explosions Large  i2mo,  3  oo 

Boyd's  Map  of  Southwest  Virginia Pocket-book  form,  2  oo 

Resources  of  Southwest  Virginia 8vo,  3  oo 

Crane ' s  Gold  and  Silver.     (In  Press.) 

Douglas's  Untechnical  Addresses  on  Technical  Subjects I2mo,  I  oo 

Eissler's  Modern  High  Explosives 8vo  4  oo 

GoesePs  Minerals  and  Metals :     A  Reference  Book i6mof  mor.  3  oo 

Ihlseng's  Manual  of  Mining 8vo,  5  oo 

*  Iles's  Lead-smelting i2mo,  2  50 

Miller's  Cyanide  Process i2mo,  i  oo 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo,  2  oo 

Peele's  Compressed  Air  Plant  for  Mines.     (In  Press. ) 

Riemer's  Shaft  Sinking  Under  Difficult  Conditions.     (Corning  and  Peels) . .  .8vo,  3  oo 

Robine  and  Lenglen's  Cyanide  Industry.     (Le  Clerc.) 8vo,  4  oo 

*  Weaver's  Military  Explosives 8vo,  3  oo 

Wilson's  Chlorination  Process limo,  i  50 

Cyanide  Processes i2mo,  i  50 

Hydraulic  and  Placer  Mining.     2d  edition,  rewritten i2mo,  2  50 

Treatise  on  Practical  and  Theoretical  Mine  Ventilation i2mo,  i  25 

SANITARY  SCIENCE. 

Association  of  State  and  National  Food  and  Dairy  Departments,  Hartfora  Meeting, 

1906 8vo,  3  oo 

Jamestown  Meeting,  1907 8vo,  3  oo 

*  Bashore's  Outlines  of  Practical  Sanitation 12mo,  i  25 

Sanitation  of  a  Country  House 12mo,  i  oo 

Sanitation  of  Recreation  Camps  and  Parks 12mo,  i  oo 

Folwell's  Sewerage.  (Designing,  Construction,  and  Maintenance.; 8vo,  3  oo 

Water-supply  Engineering 8vo,  4  oo 

Fowler's  Se\»age  Works  Analyses 12mo,  2  oo 

Fuertes's  Water-filtration  Works 12mo,  2  50 

Water  and  Public  Health 12mo,  i  50 

Gerhard's  Guide  to  Sanitary  House-inspection 16mo,  i  oo 

*  Modern  Baths  and  Bath  Houses   8vo,  3  oo 

Sanitation  of  Public  Buildings 12mo,  i  50 

Hazen's  Clean  Water  and  How  to  Get  It Large  I2mo,  i  50 

Filtration  of  Public  Water-supplies 8vo,  3  oo 

Kinnicut,  Winslow  and  Pratt's  Purification  of  Sewage.     (In  Press. ) 

Leach's   Inspection   and    Analysis  of  Food  with  Special  Reference   to  State 

Control 8vo,  7  oo 

Mason's  Examination  of  Water.     (Chemical  a::d  Bacteriological) 12mo,  i  25 

Water-supply.  (Considered  principally  from  a  Sanitary  £tandpoint) .  .  8vo,  4.  oo 
18 


*  Merriman's  Elements  of  Sanitary  Engineering 8vo,  a  oo 

Ogden's  Sewer  Design I2mo,  2  oo 

Parsons's  Disposal  of  Municipal  Refuse 8vo,  2  oo 

Prescott  and  W.inslow's  Elements  of  Water  Bacteriology,  with  Special  Refer- 
ence to  Sanitary  Water  Analysis I2mo,  50 

*  Price's  Handbook  on  Sanitation 12mo,  50 

Richards's  Cost  of  Food.     A  Study  in  Dietaries 12mo,  oo 

Cost  of  Living  as  Modified  by  Sanitary  Science I2mo,  oo 

Cost  of  Shelter 12mo,  oo 

*  Richards  and  Williams's  Dietary  Computer 8vo,  50 

Richards  and  Woodman's  Air,  Water,  and  Food  from  a  Sanitary  Stand- 
point  ; 8vo,  2  oo 

Rideal's  Disinfection  and  the  Preservation  of  Food Evo,  4  oo 

Sewage  and  Bacterial  Purification  of  Sewage .8vo,  4  oo 

Soper's  Air  and  Ventilation  of  Subways.     (In  Press.) 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  oo 

Venable's  Garbage  Crematories  in  America 8vo,  2  oo 

Method  and  Devices  for  Bacterial  Treatment  of  Sewage 8vo,  3  oo 

Ward  and  Whipple's  Freshwater  Biology.     (In  Press.) 

Whipple's  Microscopy  of  Drinking-water 8vo,  3  50 

*  Typhod  Fever Large  I2mo,  3  oo 

Value  of  Pure  Water Large  I2mo,  i  oo 

Winton's  Microscopy  of  Vegetable  Foods 8vo,  7  50 

MISCELLANEOUS. 

Emmons's  Geological  Guide-book  of  the  Rocky  Mountain  Excursion  of  the 

International  Congress  of  Geologists Large  8vo,  i  50 

Ferrel's  Popular  Treatise  on  the  Winds 8vo,  4  oo 

Fitzgerald's  Boston  Machinist i8mo,  i  oo 

Gannett's  Statistical  Abstract  of  the  World 24mo,  75 

Haines's  American  Railway  Management 12mo,  2  50 

*  Hanusek's  The  Microscopy  of  Technical  Products.     (Winton) 8vo,  5  oo 

Ricketts's  History  of  Rensselaer  Polytechnic  Institute   1824-1894. 

Large  12 mo,  3  oo 

Rotherham's  Emphasized  New  Testament , Large  8vo,  oo 

Standage's  Decoration  of  Wood,  Glass,  Metal,  etc 12mo,  oo 

Thome's  Structural  and  Physiological  Botany.     (Bennett) 16mo,  25 

Westermaier's  Compendium  of  General  Botany.     (Schneider) 8vo,  oo 

Winslow's  Elements  of  Applied  Microscopy 12mo,  50 


HEBREW  AND  CHALDEE  TEXT-BOOKS. 

Green's  Elementary  Hebrew  Grammar i2mo,     i  25 

Gtsenius's  Hebrew  and  Chaldee  Lexicon  to  the  Old  Testament  Scriptures. 

(Tregelles.) Small  4to,  half  morocco,    5  oo 

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